protein folding and dynamics of calmodulin via f-nmr · calmodulin (cam) is a ubiquitous calcium...

Protein Folding and Dynamics of Calmodulin via 19F-NMR

by

William Thach

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Biochemistry University of Toronto

© Copyright by William Thach 2012

ii

Protein Folding and Dynamics of Calmodulin via 19F-NMR

William Thach

Master of Science

Department of Biochemistry

University of Toronto

2012

Abstract

Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of

enzymes involved in cell signaling pathways. In its calcium loaded state, CaM is extremely

resistant to heat denaturation, with a melting temperature (Tm) of around 115°C. In this study,

Xenopus laevis CaM was prepared such that the eight phenylalanine residues were substituted

with 3-fluorophenylalanine. 19

F NMR studies then focused on properties of the hydrophobic core

associated with the folding process at temperatures near the regime where the protein is

completely folded. Near 70°C, near-UV circular dichroism and 1H NMR-based measurements of

protein diffusion rates reveal the onset of a stable, expanded near-native folding intermediate. 19

F

NMR solvent isotope shifts reveal a gradual loss of water from the hydrophobic core with

increasing temperature, until the point at which the near-native intermediate state is attained. At

this point, water is observed to enter the hydrophobic core and destabilize the protein.

Paramagnetic shifts from dissolved oxygen reveal an increase in oxygen accessibility with

temperature until the near-native intermediate is reached, whereupon oxygen solubility

decreases. Taken together, we conclude that hydrophobicity of the protein interior increases with

temperature, until a dry near-native state is established, whereupon water cooperatively enters

and destabilizes the hydrophobic core. 19

F CPMG experiments provide a measure of the

interconversion between the folded state and the dry near-native intermediate; at higher

iii

temperatures, folding rates are on the order of 10,000 Hz. Moreover, as temperature is lowered,

folding rates increase, presumably because the effect of off-pathway misfolding events on the

exchange process is diminished.

iv

Acknowledgments

I would like to start off by saying it has been a wonderful experience at the University of

Toronto. I am ecstatic that in the end, I was able to continue to reach and sustain high levels of

success once again. The completion of this thesis is of course a very special moment that I get to

share with all of my mentors, friends and family.

I need to start by thanking my supervisor, Dr. Scott Prosser whose guidance, support and

leadership have been instrumental in my development as a research scientist. Plus, I must admit

his great style and brilliant sense of humour make him my role model. I especially enjoyed

interacting with his dog Drifter, who shares my passion for tennis balls.

Throughout my time at U of T, I have had the opportunity to interact with so many talented and

interesting individuals. I have to thank the members of the Prosser lab: Sacha Larda, Rohan

Alvares and Tae-Hun Kim for providing me with an enjoyable lab environment. I greatly

appreciated the work that Joshua Hoang did when he worked with me for a year as an

undergraduate student. I would like to thank Sameer Al-Abdul Wahid for reinforcing my self-

belief not only as a scientist but also as a person. I would like to recognize Dr. Juli Kitevski-

LeBlanc for providing a strong experimental foundation that I was able to build upon. A big

thank you as well to the Kanelis and MacDonald research groups for sharing their resources

selflessly.

I was extremely fortunate and grateful to have Dr. Julie Forman-Kay and Dr. Simon Sharpe on

my committee. I have received incredibly helpful advice and suggestions from the discussions

about my studies. To have had the perspective of such talented and experienced research

scientists is truly an honour.

I would like to thank my dearest friends and my family who support and trust me in any situation

but also respect me enough to give me their honest opinions. I appreciate each and every one of

you and I look forward to making more memories together over the years.

Finally, I would like to end on a philosophical note. Anyone who knows me knows that tennis is

near and dear to my heart. A passage from my favourite poem, “If” by Rudyard Kipling is

written on the walls of Centre Court Wimbledon just above the entrance: “If you can meet with

v

triumph and disaster. And treat those two imposters just the same.” it reads. This excerpt has held

important meaning to me over the years. It reminds me to keep things in perspective and to carry

on with an air of dignity even in the face of the toughest challenges. It reminds me that as

insatiable as my appetite for winning is, winning doesn’t define success. For me I’ve come to

learn that it’s about the journey and the process. It’s about knowing that I’ve done as much as I

could to improve. And most importantly, it’s about being healthy and happy. This has been a key

to my success which has served me well over the years and may be of use to you. At the very

least I hope that you have been enlightened by this short aside.

vi

Table of Contents

Abstract ...................................................................................................................................... ii

Acknowledgments ..................................................................................................................... iv

Table of Contents ...................................................................................................................... vi

List of Tables ............................................................................................................................ ix

List of Figures ............................................................................................................................ x

List of Appendices................................................................................................................... xiii

Chapter 1 Literature Review ....................................................................................................... 1

1.1 Thesis Overview ............................................................................................................. 1

1.2 Calmodulin ..................................................................................................................... 2

1.3 Protein Folding with Dry Intermediate ............................................................................ 3

1.4 Calmodulin Folding Pathway .......................................................................................... 5

Chapter 2 19

F NMR & Biosynthetic Labeling .............................................................................. 6

2.1 Introduction .................................................................................................................... 6

2.2 Traditional NMR Studies ................................................................................................ 6

2.3 19F NMR and Fractional Labeling ................................................................................... 8

2.4 Assessment of Structural Perturbation of CaM Due to Fractional Labeling ................... 10

Chapter 3 19

F NMR Studies of Protein Folding ......................................................................... 11

3.1 Introduction .................................................................................................................. 11

3.2 Materials and Methods .................................................................................................. 11

3.2.1 Protein Expression............................................................................................. 11

3.2.2 Protein Purification ........................................................................................... 12

3.2.3 Trifloroethanol (TFE) ........................................................................................ 13

3.2.4 NMR Experiments............................................................................................. 14

3.3 Results & Discussion .................................................................................................... 14

vii

3.3.1 Chemical Shift................................................................................................... 14

3.3.2 Line Width as a Function of Temperature .......................................................... 16

3.3.3 Line Width as a Function of Temperature with 5% TFE .................................... 17

3.3.4 Solvent Isotope Shifts ........................................................................................ 17

3.3.5 Paramagnetic Oxygen Shifts .............................................................................. 19

3.3.6 Local Hydrophobicity ........................................................................................ 21

3.4 Conclusions .................................................................................................................. 22

Chapter 4 Circular Dichroism ................................................................................................... 24

4.1 Introduction .................................................................................................................. 24

4.2 Circular Dichroism ....................................................................................................... 24

4.3 Materials and Methods .................................................................................................. 25

4.4 Results and Discussion.................................................................................................. 25

4.5 Conclusions .................................................................................................................. 26

Chapter 5 Diffusion .................................................................................................................. 28

5.1 Introduction .................................................................................................................. 28

5.2 Pulse Field Gradient Stimulated Echo ........................................................................... 28

5.3 Results and Discussion.................................................................................................. 29

5.4 Conclusions .................................................................................................................. 30

Chapter 6 Quantitative 19

F NMR Studies of Protein Folding Dynamics .................................... 31

6.1 Introduction .................................................................................................................. 31

6.2 Carr-Purcell-Meiboom-Gill (CPMG) Relaxation Dispersion ......................................... 31


6.3.1 19F CPMG Experiments ..................................................................................... 33

6.3.2 CPMG with the Addition of TFE to 5% ............................................................. 36

6.4 Conclusions .................................................................................................................. 37

Chapter 7 19

F NMR Studies of Trypsinized CaM Fragments TR1C and TR2C .......................... 38

viii

7.1 Introduction .................................................................................................................. 38

7.2 Trypsin Fragments of Calmodulin ................................................................................. 38

7.3 Polyvinylpyrrolidone as a Molecular Crowding Additive .............................................. 39

7.4 Materials & Methods .................................................................................................... 39

7.4.1 Trypsin Cleavage .............................................................................................. 39

7.4.2 Purification of TR1C and TR2C ........................................................................ 39


7.5.1 19F Spectra of TR1C and TR2C ......................................................................... 40

7.5.2 19F CPMG Experiments ..................................................................................... 40

7.5.3 CPMG on TR1C with the Addition of TFE to 5% (v/v) ..................................... 45

7.5.4 Addition of PVP to TR2C.................................................................................. 45

7.6 Conclusions .................................................................................................................. 46

Chapter 8 Final Conclusion and Future Directions .................................................................... 47

8.1 Conclusions .................................................................................................................. 47

8.2 Future Directions .......................................................................................................... 49

References ................................................................................................................................ 52

Appendix A-Full-Length CaM Dispersion Curves by Residue .................................................. 55

Appendix B-TR1C CaM Dispersion Curves by Residue ........................................................... 56

Appendix C-TR2C CaM Dispersion Curves by Residue ........................................................... 57

ix

List of Tables

Table 1 Native and Labeled CaM EF-Hand Ca2+

Binding Constants 10

Table 2 Extracted Exchange Rates from the 19

F CPMG Dispersion Profiles of 35

3-FPhe CaM



3-FPhe TR2C CaM



3-FPhe TR1C CaM

x

List of Figures

Chapter 1

Figure 1 X-ray Structure of Calmodulin (PDB file 1CLL) 2

Figure 2 Rugged Energy Landscape Folding Funnel 4

Chapter 2

Figure 3 15

N-1H HSQC of 70% 3-

19F-Phe fractionally labeled CaM as a function 7

of temperature.

Figure 4 15

N-1H CPMG spectra of 70% 3-

19F-Phe Fractionally Labeled CaM 7

at 50°C at Three Refocusing Frequencies

Figure 5 A) X-ray structure of calmodulin (PDB file 1CLL) showing the location 9

of the eight phenylalanine residues

B) 19

F NMR spectra of calmodulin enriched with 3% to >95% 3-FPhe

Chapter 3

Figure 6 Variation in Lysozyme Structural Properties with Increasing TFE 14

Concentrations Between 0% and 50% (v/v): NOE contacts, fluorescence

λ max, and CD ellipticity at 222 nm

Figure 7 19

F NMR Spectra of 70% 3F-Phe Fractionally Labeled CaM 15

as a Function of Temperature.

Figure 8 Temperature Dependence of 19

F chemical shifts 15


F Line Widths 16


F Line Widths upon Addition of 5% 17

(v/v) 2,2,2 trifluoroethanol.

xi

Figure 11 Temperature Dependence of Normalized 19

F NMR Solvent Isotope 18

Shifts


F NMR Paramagnetic 20

Oxygen Shifts

Figure 13 Temperature Dependence of Hydrophobicity Quotient 22

Chapter 4

Figure 14 Near-UV Ellipticity Spectra as a Function of Temperature. 25

Figure 15 Average Near-UV Ellipticity as a Function of Temperature: 26

A. Without TFE B. In the presence of TFE to 5% (v/v)

Chapter 5

Figure 16 Pulse Field Gradient Stimulated Echo Pulse Sequence 29

Figure 17 Hydrodynamic radius Temperature Dependence 30

Chapter 6

Figure 18 CPMG Pulse Sequence 32

Figure 19 General Relaxation Dispersion Curve 32

Figure 20 19

F CPMG Relaxation Rate Dispersion Profiles at 45C 33

Figure 21 19


Figure 22 19


Figure 23 Exchange Rates of Full-Length CaM as a Function of Temperature 36

Figure 24 19

F CPMG Relaxation Rate Dispersion Profiles of Full-length CaM 36

with 5% TFE (v/v) at 57C.

xii

Chapter 7

Figure 25 A. Solution NMR Structure of TR1C Calmodulin (PDB file 1AK8) and 38

B. X-ray Structure of TR2C Calmodulin (PDB file 1FW4)

Figure 26 Stacked 1D 19

F spectra 40

Figure 27 19

F CPMG Relaxation Rate Dispersion Profiles of TR1C at 35C 41

Figure 28 19


Figure 29 19


Figure 30 19


Figure 31 19


Figure 32 19


Figure 33 Exchange rates of CaM as a Function of Temperature 44

Figure 34 19

F CPMG Relaxation Rate Dispersion Profiles of TR2C CaM 45

with 5% TFE (v/v) at 57C

Figure 35 19

F CPMG Spectra of 70% 3-19

F-Phe Fractionally Labeled CaM 46

at 50°C at Three Refocusing Frequencies

Chapter 8

Figure 36 Model of Hydrophobic Core Desolvation and Solvation 48

Figure 37 X-ray structure of A. Apo-calmodulin (PDB file 1CFD) 50

B. Holo-Calmodulin (PDB file 1CLL)

xiii

List of Appendices

Appendix A-Full-Length CaM Dispersion Curves by Residue 55

Appendix B-TR1C CaM Dispersion Curves by Residue 56

Appendix C-TR2C CaM Dispersion Curves by Residue 57

1

Chapter 1 Literature Review

1.1 Thesis Overview

The primary objective of the work presented in this thesis is the application of 19

F NMR to the

study of folding process along the temperature unfolding pathway. We will explore changes in

the hydrophobic core of CaM with temperature through the measurement of solvent exposure,

oxygen penetration and hydrophobicity using 19

F NMR. Subsequently, the dynamics involved in

this process will be quantitatively measured using 19

F CPMGs. This thesis is organized into the

following chapters:

(1) Introduction: This section begins with a review of calmodulin structure and function,

followed by a survey of protein folding fundamentals. Finally, calmodulin protein folding is

discussed.

(2) 19

F NMR & biosynthetic labeling: We begin by presenting CaM studies using traditional

(H,C,N) NMR experiments. Next we give a description of the properties of the fluorine nucleus

highlighting its utility and advantages in protein studies. This is followed by a discussion on

fractional protein labeling. Lastly, control experiments and results are presented to ensure that

the protein has not been excessively perturbed.

(3) 19

F NMR studies of protein folding: 19

F chemical shifts, line widths, solvent isotope shifts,

and paramagnetic oxygen shifts are evaluated along a temperature destabilization pathway to

attain details on changes in to the hydrophobic core of CaM due to the transition between a

native and near-native state.

(4) Circular dichroism: Near-UV CD measurements are made to evaluate the tertiary structure of

CaM along a temperature destabilization pathway.

(5) Diffusion: Diffusion measurements along a temperature series are made using 1H NMR

measurements. The analysis of this data allowed us to obtain the hydrodynamic radius with

changes in temperature.

2

(6) CPMG studies on full-length CaM: The use of 19

F CPMGs allowed for a quantitative

assessment of the excursions between states, namely exchange rates both as a global process and

as a residue-specific process.

(7) CPMG studies on trypsinized CaM fragments: A trypsin digestion proteolytically cleaves

CaM into two similar fragments. Analysis of the dynamics of each fragment, via 19

F CPMGs,

allowed for an assessment of cooperativity between the domains in terms of the observed

fluctuations.

(8) The final chapter briefly summarizes the results presented and future applications are

discussed.

1.2 Calmodulin

Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of

enzymes involved in cell signalling pathways. CaM contains 148 amino acid residues and has a

molecular weight of 16.7 kDa.1 The structure of CaM consists of two terminal globular domains

connected by a flexible central linker giving the protein a “dumbbell” shape. Each globular

domain contains a pair of Ca2+

binding EF hands. The EF hand binding motifs are rich in

aspartate and glutamate residues whose negative charges form a calcium binding pocket.1

Figure 1 X-ray Structure of Calmodulin (PDB file 1CLL).

3

When calcium binds the apo-CaM state, a transition to the holo-state occurs which causes the

solvent exposure of large hydrophobic patches in the globular domains. Exposure of these

hydrophobic patches is instrumental in the binding of target proteins. These patches are rich with

methionine residues which have been shown to be flexible in recognizing variable targets with

different amino acid side chains.2 The C-terminal EF hand pair is significantly higher in affinity

for calcium compared to the N-terminal domain EF hand pair.3 Each individual EF hand has a

different calcium binding affinity and calcium binding is also cooperative within each domain. It

follows that CaM can be saturated to various degrees and this allows for CaM to adopt many

conformations.4 The degree of calcium saturation is the basis for the flexible and dynamic nature

of native CaM and its ability to interact with over 300 different binding partners, while the

methionine plasticity affords CaM a degree of specificity in binding partners.5

1.3 Protein Folding with Dry Intermediate

Protein folding is the process by which a protein assembles into its native three-dimensional

structure starting from a disordered polypeptide chain. Currently, proteins are thought to fold

along funnel-shaped energy landscapes which describe the conformational heterogeneity of a

protein (Figure 2). At the top of the funnel there is a high density of states due to the high

conformational entropy of the unfolded polypeptide chain.6 Graphically, this is represented as a

wide opening to the funnel. If the protein were to sample the entire conformational space,

proteins would take infinitely longer than the observed folding timescales which are typically on

the order of 20-200μs for classic two-state folders.7 This observation is known as the Levinthal

paradox. The Levinthal paradox is reconciled by the fact that on fast timescales, small peptide

fragments search for native-like contact containing metastable structures simultaneously. These

local metastable structures then interact with each other in such a way that the native complexity

of the structure increases, essentially guiding the protein folding process towards the native

state.7 This process is known as hierarchical protein folding. Hierarchical protein folding is

analogous to a depression in the funnel from a decrease in the conformational energy and a

decrease in conformational space.6

4

Figure 2 Rugged Energy Landscape Folding Funnel.6

Thermodynamically, protein folding is primarily driven by the hydrophobic effect. The

hydrophobic effect is an entropically driven event where water molecules form ordered

structures around hydrophobic surface areas.8 This restricts the conformational and rotational

freedom of the water molecules involved. As the protein folds, the hydrophobic chains pack into

a core to release the caged water molecules which minimizes the surface area exposure of water

to the protein.9

During the folding process, kinetic intermediates can accumulate. The landscape theory thus

describes a funnel with rugged surface topography due to the establishment of these

intermediates, which may involve on-pathway and/or off-pathway folding events.7 In the final

stages of folding, a dry near-native intermediate has been proposed to be a pre-cursor to the

native state. Water molecules in the hydrophobic core are expelled to allow the protein to adopt

a dry state. This final pre-cursor is then hydrated to its final native and compact structure.10

There is evidence that as a protein unfolds, the native state initially swells into a dry molten

globule where tight packing interactions are lost but not to the degree where water can solvate

the hydrophobic core.10

The existence of this near-native folding intermediate poses several

fundamental questions. For example, what essential features of the near-native intermediate

5

facilitate the folding process? What is the role of water in stabilizing the near-native

intermediate? At what point in the process does water enter allowing for the dissolution of

specific structure? To what extent might near-native intermediate states serve a functional role,

allowing proteins to access biologically important excited states, or indeed, misfolded off-

pathway states? To study the transitions associated with the formation of the native folded state

from this intermediate, we explore changes in the hydrophobic core, primarily through 19

F NMR

along the temperature denaturation pathway.

1.4 Calmodulin Folding Pathway

Much of the research on calmodulin folding has involved single molecule studies and the use of

mechanical force to physically denature the protein. These experiments revealed a multitude of

pathways involved in the folding pathway of CaM suggesting a broad, energetically degenerate

transition state ensemble common to fast folding proteins.11

Another group using single-

molecule force spectroscopy and mechanical force as a denaturant observed a complex network

of on- and off-pathway intermediates.12

Single molecule FRET experiments have also been

performed. In these experiments, urea was used to denature CaM to study unfolding. They

discovered changes in the single-molecule distributions of the distance between fluorophores.

Their conclusion was that either a range of intermediate states or a single unfolding intermediate

existed along the urea unfolding pathway.13

Single molecule experiments have been extremely

useful in making these observations that would otherwise be lost in macroscopic measurements;

however, there is a need to correlate individual stochastic observations and ensemble or

macroscopic observations. The average behavior of individual molecules is critical to understand

as they dictate the global dynamics of the system. Ensemble experiments are advantageous in

that they are amenable to a greater number of studies than single molecule experiments.14

Furthermore, ensemble experiments can avoid harsh and unnatural physical perturbations to the

system used in the single molecule studies. Herein we describe novel biophysical methods of

studying protein folding in a minimally perturbed system. Our studies focused on the

conformational and dynamic changes associated with the hydrophobic core as a function of

temperature. We emphasize that we are able to study the dynamic properties of the hydrophobic

core as it transitions to a folding intermediate without the use of chemical or physical denaturant

or mutants.

6

Chapter 2 19F NMR & Biosynthetic Labeling

Note: Data and analysis from section 2.4 were provided by Dr. Juli Kitevski-LeBlanc

2.1 Introduction

The most commonly employed NMR experiments typically make use of 1H,

13C and

15N

resonances in a one-, two-, or three-dimensional format (experiments include 15

N-1H, or

13C-

1H

correlation spectra, plus the measurement of heteronuclear NOEs, T1, and T2 relaxation times).

These classic NMR studies are most useful for general backbone measurements and the

monitoring of large global changes, however they are typically not the most sensitive in isolating

the subtle and rapid folding and unfolding phenomena involved in transitions between states in

fast-exchange. The 19

F nucleus is a far more effective probe of Van der Waals and electrostatic

environments and hence subtle structural differences between native and near-native states.

Furthermore, we used fluorine labeled probes localized in the hydrophobic core which offer a

physical level of sensitivity to complement the enhanced chemical sensitivity. The labeling was

also shown not to perturb the system in any significant way.

2.2 Traditional NMR Studies

Traditional NMR studies such as 15

N-1H HSQCs and

15N-

1H CPMGs have been unsuccessful in

detecting an intermediate state of CaM. An HSQC is a standard protein NMR experiment which

maps all N-H correlations, the majority of which come from the backbone aminde groups.

Therefore, each residue, except proline, yields a peak in the HSQC spectra. Magnetization is first

transferred from hydrogen to attached 15

N nuclei.15

Next, the chemical shift is evolved on the

nitrogen and lastly the magnetization is then transferred back to the hydrogen for detection. From

the N-H correlation map, we can determine how folded the protein is based on how dispersed the

peaks are. More importantly information about structural or conformational changes can also be

obtained when varying experimental parameters such as temperature.15

As shown in Figure 3, a temperature titration of holo-CaM 15

N-1H HSQC spectra did not reveal

the presence of any folding intermediate, despite circular dichroism and diffusion evidence to the

contrary, as discussed later. The shifts vary linearly with temperature. Moreover, the cross peaks

7

did not show any behavior consistent with an interconversion process (i.e., change of direction in

chemical shifts and/or line broadening) and are well dispersed, indicating that the protein is fully

folded in the temperature range employed.

Figure 3 15

N-1H HSQC of 70% Fractionally Labeled holo-CaM as a Function of Temperature.

CPMGs are NMR experiments which can measure dynamic fluctuations on the millisecond time

scale (see section 6.2). Using a 15

N-1H CPMG, we observed that the

15N transverse

magnetization decay rates were independent of refocusing frequency which is indicative of the

presence of a single state as shown below.

Figure 4 15

N-1H CPMG Spectra of 70% 3-

19F-Phe Fractionally Labeled CaM at 50°C at Three

Refocusing Frequencies.

8

Evidently, these classic backbone NMR experiments either do not have the sensitivity to detect

intermediate states along the temperature unfolding pathway or no intermediate states exist.

Given that CD measurements and protein diffusion measurements point toward a subtle near-

native intermediate state around 65°C, the lack of a dispersion in the 15

N CPMG experiments

implies that backbone fluctuations are likely low in amplitude, and/or fluctuations may occur at a

rate that is too fast to detect. Our approach was to make use of 19

F NMR and corresponding 19

F

NMR CPMG relaxation dispersion measurements to discriminate subtle conformational changes

in the hydrophobic core (largely involving side chains) and access faster motions.

2.3 19F NMR and Fractional Labeling

Fluorine has a spin ½ nucleus making it amenable to NMR studies, but more importantly, there

is essentially no natural incorporation of fluorine in biological systems, 19

F exists in 100%

natural abundance, and is 83% as sensitive as 1H (cubed gyromagnetic ratio of

19F is 83% that of

1H). Therefore,

19F NMR allows for high sensitivity coupled with the virtual absence of

background fluorine signal.16

Another feature of 19

F NMR is its huge chemical shift dispersion

with changes in the local environment. The shift parameter range is hundreds of times larger than

for the 1H nuclei. This is advantageous in allowing us to sample excited states while assessing

folding and unfolding conformational exchange rates by CPMG studies; faster exchange

processes are easier to detect under circumstances where the states of interest are separated by

large frequencies.16

NMR studies of proteins often employ the use of fluorinated amino acid analogs to probe regions

of interest in the protein. It is important to perturb the structure, function and dynamics of the

natural system as little as possible. Fluorinated amino acids are thought to be essentially isosteric

with respect to the natural unfluorinated analogs. Moreover, the fluorine Van der Waals radius is

less than 20% larger than the hydrogen atom.16

In most cases, full enrichment of the biosynthetic analog is desired to prevent the accumulation

of multiple conformers leading to inhomogeneous line broadening. However, for calmodulin it

has been shown that 60-76% fractional labeling gives the ideal spectra in terms of having the

most narrow line widths and the least number of conformers.17

9

In our studies, a mono-fluorinated phenylalanine analog, 3-fluorophenylalanine was

biosynthetically incorporated into Xenopus laevis calmodulin. Calmodulin contains eight Phe

residues, five (F12, F16, F19, F65, and F68) are in the N-terminal domain and three (F89, F92,

and F141) are in the C-terminal domain. Phenylalanine residues are aromatic and hydrophobic in

nature which localizes them to the hydrophobic core upon folding. This makes them sensitive to

conformational changes during folding and unfolding events.

CaM samples were prepared with varying degrees of 3-fluorophenylalanine enrichment and it

was observed that 60-76% labeling gave the most ideal spectra. The enrichment levels were

determined by comparing fluorinated protein concentration to total protein concentration. The

fluorinated protein concentration was determined by integration of the 3F-Phe peaks relative to a

standard while the total protein concentration was determined using a BCA assay.17

Figure 5 A. X-ray structure of calmodulin (PDB file 1CLL) showing the location of the eight

phenylalanine residues. B. 19

F NMR spectra of calmodulin enriched with 3% to >95% 3-FPhe.

peak assignments were obtained previously (Kitevski-LeBlanc et al.).17

10

2.4 Assessment of Structural Perturbation of CaM Due to Fractional Labeling

It is desirable to minimally disrupt the system so that the observations from the experiments are

relevant to the native system, therefore it is important to assess any perturbations to the structure

and function of the protein caused by an alteration to the natural system. Control experiments

were done to ensure the stability and equivalence of the labeled system to that of the unlabeled

species, namely HSQCs, temperature melts and binding assays. 15N-

1H HSQCs of the ~70%

labeled protein and the native protein were virtually unchanged suggesting that the backbone

conformation was unaltered by the labeling. Temperature melts on both the labeled and native

protein were carried out by monitoring the far-CD ellipticity as a function of temperature. The

melting points for the labeled and native apo-CaM were 50.5°C ± 0.2°C and 50.8°C ± 0.2°C

respectively.18

(Note that melting points were performed on apo-CaM due to the high thermal

stability of holo-CaM.) Finally, structural perturbations were assessed from the perspective of

calcium affinity. A competitive calcium binding assay was performed through a titration of the

chromophoric chelator, 5,5’-BR2-BAPTA. The binding was monitored at OD263 nm using UV

spectroscopy until saturation was observed. Binding constants for each of the 4 EF-hands (K1-

K4) were obtained.18

Native Labeled

K1 (M-1

) 2.4 x 105 2.6 x 10

5

K2( M-1

) 1.1 x 106 1.5 x 10

6

K3 (M-1

) 3.8 x 104 8.6 x 10

4

K4 (M-1

) 1.2 x 105 1.7 x 10

5

Table 1 Native and Labeled CaM EF-Hand Ca2+

Binding Constants.17

Although there is slight variation in the binding constants, the calcium binding in general is

minimally perturbed. In conclusion, fractional labeling of CaM with 3-fluorophenylalanine

minimally perturbs the protein.

11

Chapter 3 19F NMR Studies of Protein Folding

3.1 Introduction

By using 19

F NMR in combination with site specific 19

F probes in the hydrophobic core, we are

well equipped to explore characteristics of the hydrophobic core such as solvent exposure,

oxygen penetration, and hydrophobicity.19

F shifts exhibit a quadratic temperature dependence

suggestive of the onset of a transition between states. This is corroborated by line widths which

explicitly point to a collective transition beginning near 50°C involving a fluctuation between

distinct states on a fast NMR timescale. To ascertain topological features of the protein, it is

possible to measure both solvent isotope shifts and paramagnetic shifts from dissolved oxygen

which provide perspective on solvent accessibility and hydrophobicity in the vicinity of each

probe location in the protein. Select experiments were also run with trifluoroethanol as a control.

3.2 Materials and Methods

3.2.1 Protein Expression

The open reading frame of Xenopus laevis CaM was cloned into the ampicillin resistant pET21b

vector (Novagen). Transformation of BL21 (DE3) E. coli competent cells with the plasmid DNA

was achieved through use of heat shock at 42°C for 45 seconds. A 20 mL overnight culture of

transformed E. coli BL21 (DE3) cells in LB was used to inoculate 1L of M9 media in 4 L flasks

supplemented with 10 mg of thiamine, 10 mg biotin, 100 mg ampicillin, 15 mL of 20% glucose,

1 mL of 1M magnesium sulphate and 1 mL of 0.1M calcium chloride.19

Ideal fractional 3-F-Phe labeling (70%) is achieved by the addition of D/L 3-F-Phe and D/L-

phenylalanine in a ratio of 3:2 to a final total mass of 35 mg per litre of culture. 21 mg of D/L 3-

F-Phe and 14 mg of D/L-phenylalanine were both dissolved in a minimal amount of MilliQ

water and then gently heated with vortexing until full dissolution was achieved. Each 1 L culture

was grown at 37°C, 200 rpm shaking to an O.D. at 600 nm of 0.75-0.8. Once an OD within this

range was reached, both D/L 3-F-Phe and D/L-phenylalanine solutions were added to the culture

followed by induction with IPTG to a final concentration of 1 mM. The cultures were left for 3

to 4 hours to express and the cells were then harvested by centrifugation at 7500 x g at 4°C for

40 minutes, and then stored at -20°C.

12

3.2.2 Protein Purification

Cells were partially thawed on ice and then re-suspended in lysis buffer (50 mM NaH2PO4, 300

mM NaCl, 10 mM imidazole, 1 mM PMSF, pH 8.0). 20 mL of lysis buffer were used per 1L of

culture. The lysed cells were then incubated on ice for 20 minutes with the addition of 1 mg/mL

of lysozyme followed by sonication. The lysate was then clarified by centrifugation at 15,000 g

for 20 minutes at 4°C. The resulting supernatant was incubated with Ni-NTA agarose resin

(Qiagen, Mississauga, Ontario, Canada) for 1 hour. The supernatant was allowed to flow through

the column and then a wash was performed using 10 column volumes of wash buffer (50 mM

NaH2PO4, 300 mM NaCl, 13 mM imidazole, 1 mM PMSF, pH 8.0). The protein was eluted

using 15 column volumes of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 13 mM imidazole,

250 mM PMSF, pH 8.0). A TCA precipitation was performed on the Ni-NTA elution by addition

of cold 50% TCA to a final concentration of 6% TCA followed by stirring on ice for 10 minutes.

After the incubation, the TCA precipitation was centrifuged at 6500 g for 20 minutes at 4°C. The

pellet was re-suspended in 40 mL of re-suspension buffer (50 mM Tris-base, 0.3 mM Tris-HCl,

pH 7.5). The protein was further purified using a hydrophobic interaction column composed of

phenyl sepharose. To run on the phenyl sepharose column, 1 M CaCl2 solution was added to a

concentration of 5 mM in the re-suspended TCA pellet to saturate CaM with Ca2+

and induce the

exposure of hydrophobic patches on CaM which bind to the column. This Ca2+

saturated protein

solution was then added to the phenyl sepharose column and allowed to flow through. The

column was then washed with 5 column volumes of buffer A (50 mM Tris-HCl, 1 mM MgCl2, 1

mM CaCl2, pH 7.5), 4 column volumes of buffer B (50 mM Tris.HCl, 0.5 M NaCl, 1 mM

MgCl2, 1 mM CaCl2, pH 7.5), and 3 column volumes of buffer C (50 mM Tris.HCl, 50 mM

NaCl, pH 7.5). Lastly, the protein was eluted from the resin with approximately 200 mL of

buffer D (50 mM Tris.HCl, 50 mM NaCl, 1 mM, pH 7.5) and collected in 5 mL fractions.

Absorbance readings of the fractions were taken at 280 nm on a Thermo UV-visible

spectrophotometer. Fractions of the highest absorbance were pooled together and then dialyzed

overnight against 5 L of NMR buffer (20 mM Tris-HCl, 100 mM KCl, 9 mM CaCl2, pH 8.0) at

4°C using 2 kDa MWCO dialysis tubing (Spectrum labs).19

The purified CaM was then

concentrated to at least 3 mM using 1 kDa MWCO centrifugal concentrator tubes.

13

3.2.3 Trifloroethanol (TFE)

2,2,2-trifluoroethanol (TFE) is thought to denature the tertiary and quaternary structures of

proteins while enhancing the helical structure. This is hypothesized to occur through

destabilization of hydrophobic interactions and stabilization of hydrogen bonding.20

More

recently, the role of TFE has been explored in more detail at lower concentrations. The effect of

TFE on proteins has been shown to increase internal protein contacts at low concentrations [5%-

10% (v/v)]. TFE achieves this by forming interactions with carbonyl oxygen atoms and surface

exposed hydrophobic groups. Using lysozyme as a model system, this was shown through an

increase of 2D NOESY contacts in the NH-NH region when TFE was added up to 10% (v/v)

TFE, followed by a decrease in NOESY contacts with increasing TFE relative to the amount of

contacts in the 0% TFE case. Fluorescence spectroscopy studies corroborated this finding. The

fluorescence emission spectra showed an increase in the relative intensity with increasing TFE

concentration up to 10%. This was accompanied by a downward blue-shift in the wavelength of

maximum intensity which suggested that the hydrophobic core residues were embedded to a

higher degree. At high concentrations, an upfield red-shift was observed which suggested

exposure of the tryptophan residues to an aqueous environment. Above 20% (v/v) TFE, TFE is

thought to penetrate the hydrophobic core and disrupt the hydrophobic core stability causing

denaturation of the protein and loss of tertiary and secondary structure. Finally circular dichroism

in the far-UV region showed the ellipticity was unchanged between 0% and 30% (v/v) TFE but

increased (more negative) from 30% to 50% (v/v). This suggested two structural states, one with

native helical structure around 10% (v/v) and more compact tertiary structure and another at

concentrations of TFE higher than 20% (v/v) with higher helical structure and decomposing

tertiary structure. The observations are summarized in Figure 6.20

Experiments within this

chapter and beyond will feature trifluoroethanol in a control role.

14

Figure 6 Variation in Lysozyme Structural Properties with Increasing TFE Concentrations

Between 0% and 50% (v/v): NOE contacts (–x–), fluorescence λ max (––), CD ellipticity (– –)

at 222 nm.20

3.2.4 NMR Experiments

NMR experiments were performed on a 600 MHz Varian Inova spectrometer (Agilent

Technologies, Santa Clara, CA) equipped with a 5 mm HCN/FCN triple resonance single

gradient salt tolerant cryogenic probe. All NMR samples were approximately 1-1.5 mM in

concentration and were maintained at 4°C. Line widths at half height were obtained from the

spectra using VnmrJ.

3.3 Results & Discussion

Note: Raw data from sections 3.3.1-3.3.6 inclusive were provided by Dr. Juli Kitevski-LeBlanc

3.3.1 Chemical Shift

19F Chemical shifts were analyzed as a function of temperature to determine if there was any

cursory evidence for an intermediate state. The observation for such a conclusion would involve

the presence of non-linear chemical shift changes while varying temperature.20

From the spectra,

it is clear that there is no prominent intermediate existing on the slow exchange timescale with

the folded state as shown in Figure 7. However, it is possible that an intermediate exists in fast

exchange with the folded state in which case we would expect to see a curved temperature

dependence of the fluorine chemical shift.21

15

Figure 7 19

F NMR Spectra of 70% 3F-Phe Fractionally Labeled CaM as a Function of

Temperature.

Figure 8 shows the temperature dependence of the phenylalanine shifts, after differencing them

from their respective shifts at 20°C. The majority of the shifts do not display a linear trend with

increasing temperature suggesting the likelihood of an intermediate state within this temperature

regime.


F Chemical shifts, , Associated With the

Phenylalanines of CaM (taken as a difference to 20°C). Data were fit to a third order polynomial

centered at 51° C.

16

3.3.2 Line Width as a Function of Temperature

Peak line widths are reporters of partially folded and unfolded states. The line widths provide a

measure of conformational heterogeneity.22

Broadening of peaks occurs as a result of

interconverting conformations in the millisecond-microsecond intermediate exchange regime

which cause the fluorine probe to sample different chemical environments and results in

differences in the observed chemical shift. As the populations of one state are shifted to the next,

line broadening occurs.22

1D 19

F spectra were taken as a function of temperature and line widths

were then obtained for each probe by deconvolution followed by fitting individual resonances to

Lorentzian functions (Figure 9). While narrowing of line widths is expected with temperature

due to motional averaging from whole body tumbling, the onset of line broadening at 50°C is

evidence of chemical exchange between two or more states. Moreover, because all 19

F

resonances exhibit the onset of exchange broadening at the same temperature, we hypothesize

that the transition may represent a cooperative process.


F Line Widths. The solid lines in the figure are the result

of a global fit, in which we assume a single transition temperature of 50°C. Data were fit to a

third order polynomial centered at 51°C.

17

3.3.3 Line Width as a Function of Temperature with 5% TFE

By the addition of TFE to the CaM sample (5% v/v), a compaction of the protein occurs which

tightens and strengthens the hydrophobic core. In an identical experiment to the one used in

3.3.2, the temperature dependence of 19

F line widths was analyzed. We observed a collective

delay in the onset of hydrophobic core fluctuations on the order of 20-25°C. We can therefore

conclude that the fluctuations that have been observed involve the hydrophobic core.20

Figure 10 Temperature Dependence of

19F Line Widths upon Addition of 5% (v/v) 2,2,2

trifluoroethanol. The vertical dashed line designates an approximate transition temperature

whereupon the majority of resonances begin to exhibit exchange broadening. Data points for

residues 89, 68 and 65 were fit to a second order polynomial centered at 71°C, while data points

for 12, 16, and 19 were fit to a third order polynomial centered at 71°C.

3.3.4 Solvent Isotope Shifts

The fluorine shielding parameter is significantly more sensitive to sample conditions than the

proton shielding parameter based on the NMR spectroscopic properties of the 19

F nucleus

described earlier.16

The observed shielding parameter depends on the following: local electron

motion near the NMR active nuclei, non-local electron motion, electric fields (from charged and

polar groups) in the solvent or protein, hydrogen bonding and short-range contacts. While the

shielding parameter is affected by the same factors regardless of the nuclei, the effects on

fluorine are significantly more pronounced compared to proton, making fluorine more sensitive

to changes in the local environment.15

When solvent H2O is exchanged for D2O, slight

differences in the dielectric constant of H2O and D2O result in chemical shift differences of

18

solvent exposed 19

F nuclei. These chemical shift differences, expressed in terms of solvent

isotope shifts, are proportional to the solvent exposed surface area and can be as large as 0.25

ppm.23

In practice, 1D 19

F NMR spectra are acquired under identical conditions and parameters with the

exception of the solvent composition. The solvent isotope shift is then reported as Δδ = δ(D2O)-

δ(H2O). Additionally, if an internal standard is used, a normalization of the observed effect to

that of a fully solvent exposed species can be undertaken where Δδ* = δ(D2O)protein-δ(H2O)protein/

δ(D2O)standard-δ(H2O)standard.24

The solvent isotope shift can be used to assess the solvent exposure of specific residues in

proteins. In our experiments, the fluorine probes reside inside the hydrophobic core under fully

folded conditions. We observed that at 30°C, the majority of the 3-flurophenylalanine probes

displayed normalized shifts that were 25-55% of the fully solvent exposed 4-fluorophenylalanine

standard which suggests that the hydrophobic core is significantly less solvent exposed than the

standard. In regards to the temperature dependence of the majority of the solvent isotope shifts,

as temperature is increased, the solvent isotope shifts decrease indicating a reduction of water in

the hydrophobic core (Figure 11).


F NMR Solvent Isotope Shifts, Δδ*(H2O).

Data were fit to a third order polynomial centered at 55.7°C.

19

The Gibbs free energy of the dehydration of the hydrophobic core must be less than 0 and

therefore favourable for this process to occur. At low temperature, the bulk water is highly

structured as an extensive hydrogen bonding network.25

An increase in temperature causes an

increase in enthalpy to the system. This effectively breaks the H-bonding network leading to an

increase in the disorder of the bulk solvent and protein vibrational dynamics. The expectation is

that the entropy of the system increases with heat.26

Water inside the hydrophobic core is more

ordered in that the molecules form cage-like structures around the non-polar residues comprising

the core but at higher temperatures an enthalpic input is needed to form these clathrates,

therefore this also results in a loss in order. These two effects in tandem allow for water to exit

the core into the bulk solvent. This trend is reversed at ~60°C, the same temperature which was

observed for the on-set of line broadening. Above 60°C, solvent isotope shifts collectively

increase suggesting a cooperative hydration of the hydrophobic core. A loss in hydrophobic

integrity in principle allows water to more freely enter the core to fill the local void volumes.

Note that two of the residues (F12 and F141) exhibit negative solvent isotope shifts. A local

structural change may have occurred as a result of the solvent exchange of H2O and D2O with

the result being that the probes may be reporting on factors other than solvent exposure. The

trend of these residues with temperature however would be expected to be independent of the

structural change.

3.3.5 Paramagnetic Oxygen Shifts

Paramagnets are species which have non spin-paired electrons and thus, are affected by magnetic

fields. They have strong effects on the relaxation (paramagnetic resonance enhancement) and the

chemical shift (pseudocontact chemical shift) of nearby nuclei.27,28

The fluorine nucleus is very

sensitive to both dipolar and contact mechanisms of interaction as a result of its large

gyromagnetic ratio and highly polarizable electrons.

Freely diffusing paramagnets such as molecular oxygen, O2, have been used to gain perspective

on chemical and physical properties NMR active probes. Oxygen is an ideal paramagnet additive

for our studies because of its small size and short electronic spin relaxation time.27,28

Its size

allows it to partition into protein void volumes and across water-membrane interfaces. In

particular, it can freely penetrate into the hydrophobic core and act as a reporter of

hydrophobicity and density of the protein interior. The short electronic spin relaxation time

20

results in minimal line broadening and substantial paramagnetic shifts. Furthermore O2 is trivial

to add or remove from a sample.29

To introduce molecular oxygen to the sample, a 1.5-2 mM CaM sample was placed in a 5 mm

OD, 3 mm ID sapphire NMR sample tube (Saint Gobain – Saphikon Crystals, Milford, NH,

USA). The sample was then equilibrated at a partial pressure of 25 atm for two days outside the

magnet followed by an overnight equilibration in the magnet. The pressure was maintained using

a pressurized oxygen supply and Swagelok connections (Swagelok, Solon, OH, USA). In our

experiments, paramagnetic shifts were measured as a function of temperature. Additionally, an

internal standard (4-fluorophenylalanine) was used to obtain a normalization of the observed

effect to that of a fully solvent exposed species where Δδ*(O2) = δ(O2)protein-δ(No O2)protein/

δ(O2)standard-δ(No O2)standard.

Figure 12 Temperature Dependence of Normalized

19F NMR Paramagnetic Oxygen Shifts,

Δδ*(O2). Data were fit to a fourth order polynomial centered at 51°C.

The scale of the chemical shift differences explicitly allows us to state that oxygen preferentially

partitions into the hydrophobic core over bulk phase solvent since the normalized shifts are all

larger than 1. In terms of the trend, there is a collective and rapid paramagnetic shift increase

with temperature increase from 20-60°C which suggests that as we heat the protein, we are

increasing the ability of oxygen to enter the hydrophobic core (Figure 12). It follows that the

core is becoming more hydrophobic over this temperature range. In conjunction with the

observations from the solvent isotope shifts, we can conclude that local void volumes caused by

21

water leaving the core are providing more opportunities for oxygen solubilization inside the core.

This trend reverses at approximately 65°C, where dissolved oxygen is now being displaced by

water in a collective hydration of the core. In considering the scale of both solvent isotope and

paramagnetic effects, we note that the amount of oxygen entry is significantly higher than the

amount of water exiting.

3.3.6 Local Hydrophobicity

Consolidating the solvent isotope shifts and paramagnetic shifts into one term effectively

represents an estimate of the partitioning potential of oxygen and water, thereby providing a

hydrophobicity index. The methodology first involves defining the contact shift from dissolved

oxygen as

Δδ(O2) = k <Ω> α<[O2]>local

where k represents a proportionality constant, <Ω> is the collisionally accessible surface area, α

represents a polarization or spin-delocalization term, and< [O2] >local represents the local oxygen

concentration.29

This definition considers only variables which give rise to the variation in shifts

from a probe within a protein. The normalized shift Δδ*(O2) gives the following ratio:

Δδ*(O2) =

The exposed surface area is expected to be the dominant factor in the contact shift, due to the

short range nature of the effect.

Similarly, the normalized solvent isotope shifts are as follows:

Δδ*(H2O) =

=

The chemical shift when D2O is exchanged with H2O is also assumed to rely on exposure of the

surface area. Given that the collisional accessibilities of water and oxygen are similar we

approximate them as being equal and consider the ratio of the normalized shifts to give:

=

22

This ratio provides an estimate of the partitioning potential of oxygen and water which we term

as the hydrophobicity index.29

Figure 13 Temperature Dependence of Hydrophobicity Quotient, Δδ*(O2)/Δδ*(H2O),

determined from experimentally determined paramagnetic shifts and solvent isotope shifts. Note

that the solvent isotope shifts of F12 and F141 have been renormalized by adding a constant to

each solvent isotope shift such that such that Δδ*(H2O) = 0.3 (the average of the remaining

phenylalanines). Data were fit to a third order polynomial centered at 55.7°C.

For the majority of probes, we observe a pronounced increase of hydrophobicity within the

hydrophobic core as we increase temperature to approximately 65°C at which point it begins to

decrease.

3.4 Conclusions

Through this first set of experiments, we have amassed strong evidence for the presence of a dry

near-native intermediate that forms through a hierarchy of events. 19

F chemical shift analysis

gave an initial indication of an intermediate as the temperature dependence was non-linear.

Through line width analysis, we have observed the presence of interconverting conformations

when a temperature of ~55°C is reached. With the addition of TFE to 5%, the onset to the

intermediate state is delayed by approximately 25 °C, which suggests that the intermediate state

involves conformational or dynamic changes of the hydrophobic core. The solvent isotope shifts

revealed that the hydrophobic core becomes desolvated as the protein is heated until ~60°C, at

which point a shift towards a more solvated state occurs. In a parallel observation, oxygen enters

23

the hydrophobic core in increasing amounts as we raise the temperature until ~60°C at which

point the dissolved oxygen is displaced by water, in other words a solvation of the hydrophobic

core occurs. The hydrophobicity index consolidates the solvent isotope and paramagnetic shift

observations. We conclude from the hydrophobicity index that the hydrophobic core increases in

hydrophobicity as we increase temperature to ~65°C but above this temperature point, the

hydrophobicity decreases. This is in line with the solvent isotope shift observation of solvation

about 60°C.

24

Chapter 4 Circular Dichroism

4.1 Introduction

Much of the data thus far has involved NMR based techniques. In this next chapter, circular

dichroism measurements were made to probe the tertiary structure with and without the presence

of 5% (v/v) TFE. The measurements were done as a function of temperature on 70% 19

F

fractionally labeled CaM.

4.2 Circular Dichroism

Circular dichroism (CD) is indispensible as a tool in evaluating the structure of proteins in

solution. CD signals are only observed when there is absorption of radiation. Distinct structural

elements therefore have characteristic regions in which they appear. For example, in proteins,

absorption below 240 nm corresponds to the peptide bond, absorption in the range 260 to 320 nm

corresponds to aromatic amino acid side chains and absorption centered around 260 nm

correspond to disulphide bonds.30

Note that chromophores of the same type in close proximity,

absorb together as a single exciton resulting in characteristic spectral features.

Aromatic side chain residues elicit CD signal within the near-UV region in the range 260-320

nm. Each of the amino acids has a unique wavelength profile. Trp shows a peak close to 290 nm

with fine structure between 290 and 305 nm, Tyr shows a peak between 275 and 282 nm, with a

shoulder at longer wavelengths often obscured by bands due to Trp, and Phe shows weaker but

sharper bands with fine structure between 255 and 270 nm.30

The shape and magnitude of the near UV CD spectra of a protein is determined by a number of

factors, including the compactness of the protein and the local environment (hydrogen bonding,

polar groups). The compactness and density of the protein influences the spectra where the more

compact and/or dense a protein is, the less mobile the side chains are rendered. This strengthens

the hydrophobic contacts within the hydrophobic core and this leads to greater signal. A less

compact and/or less dense protein conformation would result in the side chains being more

mobile, thus lowering the observed intensity of the signal.31

25

4.3 Materials and Methods

Near-UV CD spectra were acquired on an Aviv CD spectrometer model 62DS at from 20C to

100C. Spectra of 2 mg/mL 70% fractionally labeled 19

F CaM were taken in 0.1 M KCl and 20

mM Tris, 9mM CaCl2 buffer adjusted to pH 8. The ellipticity was measured and collected from

253 nm to 273 nm (path length, 0.1 cm; steps, 0.05 nm; bandwidth, 1 nm; and averaging time 0.2

s).

4.4 Results and Discussion

CaM contains two tyrosine residues and does not contain any tryptophan residues. Therefore, the

CD signal in the near-UV region is more or less exclusively attributed to the phenylalanine

residues. In our experiment, CD spectra were taken in the range 253-273 nm as a function of

temperature from 15°C to 100°C. Firstly, we can conclude that the protein is folded in a well-

defined structure due to the observation of significant near-UV signal over the temperature range

monitored. Furthermore, with a cursory look at the raw spectra, we observe that with increasing

temperature, the ellipticity tends towards zero and loses fine structure detail. This suggests

increased mobility of the hydrophobic side chains with temperature.

Figure 14 Near-UV Ellipticity Spectra as a Function of Temperature.

26

For a more concise analysis of the data, the ellipticity values at each temperature were summated

and then averaged. In figure 15A, we observe the ellipticity tending towards zero as the

temperature increases from 20°C to 60°C, indicative of a loss of compactness or density. At

60°C, we see the onset of an intermediate state whereby the ellipticity increases and reaches a

local minimum before moving towards zero once again. TFE was then added to the sample to a

final concentration of 5%. Near-UV spectra were then taken as a function of temperature and the

same data analysis was undertaken. The results show a complete abolition of the local minima

resulting from the proposed intermediate state. (Figure 15B). Presumably, the TFE will have

pushed the transition to a higher temperature as we observed with the line widths earlier (Figure

10).

Figure 15 Average Near-UV Ellipticity as a Function of Temperature: A. Without TFE. Data in

the temperature range 20-60°C was fit to a third order polynomial, uncentered, while data in the

temperature range 65-95°C was fit to a fourth order polynomial centered at 77.5°C.B. In the

presence of TFE to 5% (v/v). Data were fit to a third order polynomial centered at 52.5° C.

4.5 Conclusions

Through near-UV measurements, we were able to corroborate previous results by identifying an

intermediate state within the temperature range of 60-70°C, and favouring the equilibrium

towards the native state through the addition of TFE. The CD results explicitly display a local

minima around 65°C which is representative of an intermediate state. Furthermore the data

describe an increase in hydrophobic core side chain mobility with temperature, followed by a

further loss in hydrophobic core side chain mobility as a result of the onset of hydrophobic core

solvation. The initial increase in side chain mobility is likely representative of a density loss as

27

opposed to a gain in compactness as we will see from the diffusion measurements. The loss in

hydrophobic side chain mobility occurs concurrently with an initial amount of water entering the

hydrophobic core. Finally as we move closer towards the denaturing temperature regime, further

water entry causes an increase in side chain mobility as stabilizing side chain interactions are lost

due to the dissolution of specific structure.10

28

Chapter 5 Diffusion

5.1 Introduction

With a near-native dry intermediate state, it would be expected that the hydrodynamic radius of

the protein would be expanded relative to the native state due to loss in close packing.10

Through

the Stokes-Einstein equation, the hydrodynamic radius can be determined through diffusion

coefficient measurements. One way to measure diffusion coefficients is through the use of an

NMR experiment called a pulse field gradient stimulated echo.

5.2 Pulse Field Gradient Stimulated Echo

The stimulated echo (STE) pulsed field gradient (PFG) NMR pulse sequence for diffusion

measurements consists of three intervals, namely, the prepare, the store and the read intervals

(Figure 16). The phases of the three 90° radio frequency pulses and of the receiver are cycled to

eliminate unwanted echoes.32

A 90x° pulse is initially applied to generate transverse

magnetization in the XY plane through rotation from the Z-axis. During the first delay time, τ2

(the prepare interval), a gradient pulse of amplitude g and duration δ labels the spins according to

their position along the direction of the applied magnetic field gradient, in this case along the Z-

axis. This is thought of as an encoding or scrambling of the spins. The magnetization is then

stored as Mz which allows for longer diffusion times.33

During the third delay time (the read

interval), τ2, the second gradient pulse decodes or unscrambles according to the position along

the direction of the applied gradient. If the molecules have moved in the longitudinal direction

during the sequence, this will manifest itself as attenuation in signal of the stimulated echo.

29

Figure 16 Pulse Field Gradient Stimulated Echo Pulse Sequence.

5.3 Results and Discussion

STE PFG experiments were performed on 70% fractionally labeled 19

F CaM as a function of

temperature (g = 3.5 T/m, δ = 0.005 s, τ1 = 0.35 s, τ2 = 0.1 s, Δ = 0.45 s). By the Stokes-Einstein

law, D =

where k is the Boltzmann constant, T is the temperature in Kelvin, η is the

viscosity of the solution, D is the diffusion coefficient and RH is the hydrodynamic radius, the

diffusion coefficient of the protein is inversely proportional to the hydrodynamic radius of the

protein.34

The radius was solved for using the Stokes-Einstein law and then plotted as a function

of temperature as shown in Figure 17.

What we observe is a gradual increase in the hydrodynamic radius with temperature. As we

approach the temperature regime where we previously observed the onset of solvation of the dry

near-native state, the hydrodynamic radius begins to decrease. The increase in hydrodynamic

radius of the intermediate state is consistent with the notion of a desolvated hydrophobic core.10

30

Figure 17 Hydrodynamic Radius as a Function of Temperature. Data were fit to a fifth order

polynomial, uncentered.

5.4 Conclusions

The hydrodynamic radius measurements obtained via diffusion rates have provided further

evidence for the presence of an intermediate state at higher temperatures. They also physically

describe the intermediate in terms of size and compactness. From previous measurements, we

know that the onset of the intermediate state involves the departure of water from the

hydrophobic core. The RH measurements show an increase in the hydrodynamic radius with

temperature until approximately 43°C, whereupon a decrease in the hydrodynamic radius occurs.

We note that this temperature seems to be lower than what we would expect based on the other

measurements. The increase in RH is consistent with a desolvation of the protein pre-onset, while

the slight decrease in RH reflects a gain in compaction that is accompanied by the entry of water.

Above 60°C it is expected that the hydrodynamic radius expands due to dissolution of specific

structure caused by further water entry.

25

30

35

40

45

50

17 22 27 32 37 42 47 52 57 62

Rad

ius o

f H

yd

rati

on

(Å

)

Temperature (°C)

31

Chapter 6 Quantitative 19F NMR Studies of Protein Folding Dynamics

6.1 Introduction

The role of molecular dynamics is critical to understanding the biological processes involved in

protein folding. NMR is capable of quantitating molecular motions over a wide range of

timescales including the ms-μs timescale which protein folding processes occur under. Using a

Carr-Purcell-Meiboom Gill NMR experiment, transverse relaxation rates, R2 can be obtained.

Relaxation rates are extremely sensitive to dynamic motions on the ms-μs timescale and

furthermore, it is possible to extract the kinetics (exchange rates) of the dynamic process from

them.35

6.2 Carr-Purcell-Meiboom-Gill (CPMG) Relaxation Dispersion

Carr-Purcell-Meiboom-Gill (CPMG) NMR experiments are sensitive to excited states that are

populated to at least 0.5% relative to the ground state. These experiments utilize the fact that

exchange or motion events affect the transverse nuclear spin relaxation rates of the perturbed

regions in a protein. Critical to the effectiveness of the CPMG is differential probe evolution at

each state.35

Using a CPMG experiment, quantitative information about the dynamics of fast and

slow-exchange protein motions on the microsecond to millisecond timescale can be obtained. In

the slow-regime, populations of the states can also be acquired.36

The CPMG experiment consists of an initial 90° excitation pulse on x to generate transverse

magnetization. This is followed by a spin-echo train which consists of a delay for a time, τcp

which allows the signal to decay away due to T2*, a 180° pulse on y which refocuses the signal

and finally a delay for τcp. The spin-echo train is repeated n times for a given big Τ (bt) value.37

bt determines the length of the spin echo train and τcp effectively determines the value of n

(Figure 18).

32

Figure 18 CPMG Pulse Sequence.

If the spin exists in a single state for the time period, 2τcp where the 180° refocusing pulse is in

the centre of this interval, then the overall frequency evolution for 2τcp is zero. Conversely, if

during the 2τcp time period, the spin undergoes a stochastic transition to another state the

refocusing pulse is considered off-centre with the result being incomplete refocusing and a loss

of peak intensity.36

To evaluate the relaxation dispersion, τcp is arrayed. The refocusing is most

effective when n is small and least effective when n is large (Figure 19).

Figure 19 General Relaxation Dispersion Curve.

There was strong qualitative evidence that the 8 probes in the hydrophobic core of a CaM

molecule undergo exchange between two conformations. Each probe should therefore exist in

one of two states with stochastic jumps between the states making CaM amenable to CPMG

studies.

33


6.3.1 19F CPMG Experiments

Using 2-3 mM, 70% 19

F fractionally labeled CaM, 19

F CPMG experiments were run on a

600MHz spectrometer at three temperatures, 45°C, 51°C, and 57°C. A big Τ value (fixed

interval) of 12 ms was used. The refocusing frequency, Τcp was arrayed with the following

values: 0.0075 ms, 0.01 ms, 0.012 ms, 0.015 ms, 0.02 ms, 0.025 ms, 0.04 ms, 0.05 ms, 0.075 ms,

0.01 ms, 0.15 ms, and 0.3 ms.

The peak intensities were obtained by deconvolution followed by fitting individual resonances to

Lorentzian functions. The peak intensities were then used to calculate a transverse relaxation rate

(R2). The R2 values for each τcp value were calculated using the following relationship:

τ

, where bt is the constant-time CPMG relaxation period, Iτcp is the peak intensity

at a given refocusing frequency, τcp, and Io is the peak intensity undeterred by relaxation decay.38

The resulting dispersion profiles at three temperatures, 45°C, 51°C, and 57°C, are given below.

Figure 20

19F CPMG Relaxation Rate Dispersion Profiles at 45C. Data were fit to an

exponential one phase decay.

34

Figure 21



Figure 22



To determine the chemical shift exchange timescale, we analyzed exchange relaxation rates at

two different field strengths, 500 MHz and 600MHz. In slow exchange regimes, no field strength

dependence exists, while for fast exchange regimes, there is a quadratic field strength

dependence. The relaxation rate was determined to be proportional to the square of the field

35

strength and therefore CaM exists in a fast-dynamic regime.39

The relaxation rate data were then

fit to a two-site fast exchange regime, given by,

CPMG

ex

ex

CPMG

ex

BA k

kk

ppRR

4tanh

41

20

22

where PA and PB are populations, Δω is the chemical shift difference, kex is the exchange rate,

is the refocusing frequency, and R2 is the relaxation rate.40

Using Mathematica fitting software, kex was extracted from the fast-exchange fit. Globally fit

rates of 18000 ± 7000 Hz, 15000 ± 2700 Hz, and 11000 ± 1600 Hz were obtained at 45°C, 51°C,

and 57°C. The exchange rates show non-Arrhenius behaviour in that they decrease with

increasing temperature. Individually fit rates by residue were also computed. The rates are

tabulated below while the dispersion curves by residue are included as appendix A.

kex (Hz)

Residue 45°C 51C 57°C

89 24000 ± 15000 30000 ± 14000 15000 ± 10000

141 24000 ± 14000 18000 ± 14000 13000 ± 11000

68 10000 ± 9600 11000 ± 4400 13000 ± 4700

65 24000 ± 16000 24000 ± 14000 16000 ± 12000

19 19000 ± 10000 18000 ± 5700 9700 ± 3900

92 21000 ± 9700 16000 ± 4200 13000 ± 6800

Global Fit 18000 ± 7100 15000 ± 2700 11000 ± 1600


F CPMG Dispersion Profiles of 3-FPhe CaM.

36

Figure 23 Exchange Rates of Full-Length CaM as a Function of Temperature. Data were fit to a

straight line.

6.3.2 CPMG with the Addition of TFE to 5%

CPMGs on the full-length protein supplemented with TFE to 5% (v/v) were run at 57 °C. No

dispersion was observed. This reinforces the observations with TFE in regards to the 19

F line

width temperature dependence, where the hydrophobic core was observed to become more stable

due to interactions with TFE.

Figure 24

19F CPMG Relaxation Rate Dispersion Profiles of Full-length CaM with 5% TFE

(v/v) at 57C. Data were fit to a second order polynomial centered at 2954 Hz.

37

6.4 Conclusions

Having identified the presence of interconverting protein states, our next goal was to quantify the

molecular dynamics involved in the transition between the states. CPMG experiments were run

at three temperatures from which relaxation decay data were extracted and then fit to a fast

exchange regime. kex rates observed were on the order of 10,000-20,000 Hz with a non-

Arrhenius trend. CPMGs with TFE did not display a dispersion. This result confirmed that the

fluctuations involved dynamics of the hydrophobic core. TFE effectively stabilized the core to

such a degree that the excursion to other states was restricted.

38

Chapter 7 19F NMR Studies of Trypsinized CaM Fragments TR1C and TR2C

7.1 Introduction

Trypsin cleaves CaM in the linker region to yield two fragments, TR1C and TR2C. To explore

the possibility of cooperativity between the two domains in the folding and unfolding transition,

CPMG experiments were performed on proteolytically cleaved CaM. Furthermore, as an initial

exploration for future in-vivo studies, the effect of a molecular crowding agent,

polyvinylpyrrolidone, on the fluctuations observed through CPMGs was addressed.

7.2 Trypsin Fragments of Calmodulin

Trypic cleavage of CaM in the calcium bound state occurs at lysine-77. This gives rise to two

closely homologous fragments. The two fragments, TR1C (N-terminal half, residues 1-77), and

TR2C (C-terminal half, residues 78-148) have shown cooperative behavior in terms of calcium

binding, but non-cooperative kinetic behavior.41

TR2C contains the stronger calcium binding EF

hands and as such, holo-CaM TR2C has been shown to be more stable than holo-CaM TR1C.42

Note that TR1C has five phenylalanine residues while TR2C contains the remaining three.

Figure 25 A. Solution NMR Structure of TR1C Calmodulin (PDB file 1AK8) and B. X-ray

Structure of TR2C Calmodulin (PDB file 1FW4).43,44

39

7.3 Polyvinylpyrrolidone as a Molecular Crowding Additive

Polyvinylpyrrolidone (PVP) is a random-coil polymer commonly used to simulate in-vivo like

conditions. It is easily soluble, has protein-like properties, and metabolically inert. Moreover, it

prevents protein aggregation and proteolyic digestion.45

7.4 Materials & Methods

7.4.1 Trypsin Cleavage

Purified CaM was concentrated to 1 mM and then dialyzed against buffer A (50 mM NH4HCO3,

50 mM NaCl, pH 7.9). Trypsin (Sigma, Mississauga, Canada) was added to 1 mM CaM for a

final concentration of 0.017 mM and then incubated for 1 hour at 37°C. The trypsin digest was

then stopped by the addition of soybean trypsin inhibitor (Sigma, Mississauga, Canada) to a final

concentration of 0.017 mM followed by incubation on ice for 15 minutes.46

7.4.2 Purification of TR1C and TR2C

The digest was added to a G-50 (Pharmacia) gel filtration column (30 cm x 1.5 cm) to separate

out the cleaved protein from the un-cleaved protein. The column was run with 2 column volumes

of buffer A (50 mM NH4HCO3, 50 mM NaCl, pH 7.9), with 5 mL fraction collection occurring

at approximately 50 mL of elution. Absorbance readings of the fractions were taken at 280 nm

and the fractions of highest absorbance were pooled together. CaCl2 was added to a final

concentration of 5 mM in the pooled fractions and then loaded onto a phenyl sepharose column.

The column was then washed with 1 column volume of buffer B (50 mM Tris-HCl, 1 mM

CaCl2, pH 7.5). Elution of the TR2C C-terminal fragment was achieved by running 2 column

volumes of buffer C (2 mM Tris-HCl, 1 mM CaCl2, pH 7.5). 5 mL fractions were collected and

then their absorbances were read at 280 nm due to the presence of 2 tyrosine residues. The TR1C

N-terminal fragment was eluted by the addition of 3 column volumes of milliQ water. The

reduction in salt concentration from buffer C to milliQ water significantly weakens the

hydrophobic interactions between TR1C and the resin to allow for the elution. 5 mL fractions

were taken and absorbances were read at 258 nm, where phenylalanine absorbs.46

40


7.5.1 19F Spectra of TR1C and TR2C

Below are the 1D 19

F spectra of the TR1C, TR2C fragments with the full-length for comparison.

Figure 26 Stacked 1D 19

F Spectra. From top to bottom: Full-length, TR2C, and TR1C.

7.5.2 19F CPMG Experiments

CPMGs were once again used to assess the kinetics of the fluctuations observed as temperature

increases. It was important to address whether the fluctuations were still present in either or both

of the trypsinized fragments and if they were, whether the fluctuations were cooperative between

domains. 19

F CPMG experiments were run on a 500 MHz spectrometer at three temperatures,

35°C, 45°C, and 55°C. A big Τ value (fixed interval) of 12 ms was used. The refocusing

frequency, Τcp was arrayed with the following values: 0.005 ms, 0.006 ms, 0.0075 ms, 0.01 ms,

41

0.012 ms, 0.015 ms, 0.02 ms, 0.025 ms, 0.04 ms, 0.05 ms, 0.075 ms, 0.01 ms, 0.15 ms, and 0.3

ms.

The peak intensities and transverse relaxation rates (R2) were obtained as before to generate

relaxation dispersion curves. The resulting dispersion profiles at three temperatures, 35°C, 45°C,

and 55°C, are given below for TR1C.

Figure 27

19F CPMG Relaxation Rate Dispersion Profiles of TR1C at 35C. Data were fit to a

one phase decay. Note: residues 65 and 19 were not included due to high noise levels.

Figure 28


one phase decay.

42

Figure 29


one phase decay.

The resulting dispersion profiles at three temperatures, 35°C, 45°C, and 55°C, are given below

for TR2C.

Figure 30


one phase decay.

43

Figure 31


one phase decay.

Figure 32


one phase decay.

Using the fitting software, Mathematica, kex was extracted from the fast-exchange fit. Global

rates of both TR1C and TR2C are tabulated below at 35°C (TR2C only), 45°C, and 55°C. The

exchange rates increase with temperature and at higher temperatures, they are significantly faster

than the observed exchange rates of the full-length protein. Rates by residue are also included

below. Dispersion curves for TR1C and TR2C are found in appendix B and C respectively.

44

kex (Hz)

Residue 45C 55°C

12 39000 ± 2400 39000 ± 2400

16 30000 ± 10000 30000 ± 10000

68 14000 ± 11000 29000 ± 9900

65 16000 ± 12000 14000 ± 5400

19 6800 ± 1200 22000 ± 4600

Global Fit 7200 ± 1500 21000 ± 4300


F CPMG Dispersion Profiles of 3-FPhe TR1C

CaM.

kex (Hz)

Residue 35°C 45C 55°C

89 15000 ± 13050 14000 ± 1500 15000 ± 4300

141 4000 ± 700 19000 ± 1000 24000 ± 2500

92 18000 ± 1300 15000 ± 610 28000 ± 550

Global Fit 16000 ± 2700 16000 ± 510 28000 ± 570


F CPMG Dispersion Profiles of 3-FPhe TR2C

CaM.

Figure 33 Exchange Rates of CaM as a Function of Temperature. Data were fit to a straight line.

45

7.5.3 CPMG on TR1C with the Addition of TFE to 5% (v/v)

CPMGs on TR1C supplemented with TFE to 5% (v/v) were run at 57 °C. No dispersion was

observed. This reinforces the observations with TFE in regards to the 19

F line width temperature

dependence, where the hydrophobic core was observed to become more stable due to interactions

with TFE.

Figure 34

19F CPMG Relaxation Rate Dispersion Profiles of TR2C CaM with 5% TFE (v/v) at

57C. Data were fit to a second order polynomial centered at 3280 Hz.

7.5.4 Addition of PVP to TR2C

The PVP CPMGs were conducted on the TR2C proteolytic fragment as it has the best peak

separation and the cleanest spectra due to the domain only having three phenylalanine probes.

PVP was added to a TR2C CaM sample for a final concentration of 50 mg/mL, followed by 300

mg/mL. At each respective PVP concentration, CPMGs were performed at 55°C. No obvious

dispersion was noted for the 50 mg/mL PVP experiment. The addition of PVP to 300 mg/mL

made the signal to noise ratio significantly worse, so much that the data could not be accurately

interpreted. Experimental parameters need to be optimized to ameliorate the data.

46

Figure 35 19

F CPMG Spectra of 70% 3-19

F-Phe Fractionally Labeled CaM at 50°C at Three

Refocusing Frequencies.

7.6 Conclusions

Both TR1C and TR2C demonstrate kexs which are significantly larger than for the full-length. At

the highest temperature point studied, TR2C has a kex approximately three times as large as for

the full-length, and TR1C has a kex approximately twice as large as for the full-length. In terms

of the temperature trend, the kex for both TR1C and TR2C increase with temperature. This is in

contrast to the non-Arrhenius behavior of the full-length protein. Taken together, this suggests

that off-pathway misfolding events between opposite domains occur in the full-length protein

which manifest in a slowing of the fluctuations observed. Therefore, cooperativity was not found

to exist in the fluctuations, instead anti-cooperative behavior between the domains was observed.

47

Chapter 8 Final Conclusion and Future Directions

8.1 Conclusions

Through the diverse array of experiments presented in the preceding chapters, we have been able

not only to observe a dry near-native folding intermediate of CaM, but also physically and

chemically describe its properties. The use of various NMR techniques as well as CD revealed a

transition to a state in which secondary and tertiary structure is largely preserved. The 19

F NMR

solvent isotope shifts highlight the role of water in the near-native state. Water was explicitly

shown to exit the hydrophobic core to establish the dry intermediate state. In parallel to water

exiting the core, paramagnetic oxygen shifts showed oxygen penetrating the core. Taken together

we have a detailed microscopic perspective of both solvent exposure and hydrophobicity.

Moreover, water was explicitly shown to penetrate the hydrophobic core after the establishment

of the intermediate, a conclusion which addresses a longstanding debate in the protein folding

research field.47

These measurements all point to a transition occurring at 60-65°C.

Protein diffusion measurements allowed us to extract the hydrodynamic radius as a function of

temperature. Within the temperature range where the protein is mostly folded, the average

hydrodynamic radius increases with temperature, swelling to ~40% larger. Upon the onset of the

near-native intermediate, the hydrodynamic radius was shown to decrease with temperature. CD

revealed an increase in side chain motion with temperature. With both diffusion and CD data in

mind, the simultaneous hydrodynamic radius decrease and freedom of side chain motion increase

suggest a loss of density in the hydrophobic core, which our other data suggest is due to water

exiting and void volumes being created. Furthermore an explicit minimum is observed around

65°C which was previously found to be the temperature at which the hydrophobicity of the

hydrophobic core was at a maximum within the studied temperature range. We conclude that this

minimum represents the dry near-native intermediate state.

Finally, through CPMG measurements, we were able to measure the transition rate between

folded and dry near-native intermediate states as a function of temperature. Folding rates were

observed to be on the order of 10,000 Hz and in the case of the full-length protein, they

decreased with temperature. In contrast, cleaved CaM fragments, TR1C and TR2C showed

48

exchange rates which increased with temperature and were significantly larger than those of the

full-length protein. This suggested that misfolding occurs in the full-length protein with higher

prevalence at higher temperatures. We hypothesize that the misfolding events are inter-domain

due to the exchange rate observations we made with the trypsinized domains.

As a control, the use of TFE in line width, CPMG and CD experiments shifted the equilibrium

dramatically to the folded state, highlighting the fact that the hydrophobic core plays a major role

in the excursions between states.

The following is a model which summarizes the observations presented in this thesis, with the

middle structure being that of the dry near native intermediate.

Figure 36 Model of Hydrophobic Core Desolvation and Solvation.

49

8.2 Future Directions

The research presented in this thesis demonstrates the applicability of a number of novel

biophysical measurements. We have been able to observe subtle changes in protein conformation

that occur on extremely fast timescales through the use of 19

F NMR and directed labeling. A

logical step to take would be to extend these novel biophysical techniques to other systems;

systems with established molten globules states would be of particular interest such as

cytochrome C.48

There are a number of interesting aspects to calmodulin that we would still like to establish

however. One field we would like to investigate is in-cell NMR. In-cell NMR involves injection

of isotopically labeled protein into living cells. This study allows us to study the conformation

and function of proteins under intracellular environments which is useful when we consider that

proteins are not trapped in one conformation but dynamically change their structure in-cell via

protein-protein interactions.49

First, we would like to refine the recent PVP CPMG experiments.

PVP acts as a molecular crowding agent in simulations of in-cell conditions. Theory has

predicted that molecular crowding stabilizes proteins.50

In our current work, temperature denaturation has been used to study the unfolding process;

however there are a great many other denaturation methods available. Pressure NMR studies are

of particular interest to us as proteins are known to undergo pressure denaturation but little is

known about the process.51

Given that pressure is a fundamental thermodynamic variable, it is

expected that pressure NMR studies could yield novel information on the folding process.

Specific volume changes would be expected to illicit changes to the fluctuations we observed by

compacting the hydrodynamic radius. Pressure is also easily adjusted using a high-pressure

NMR tube and apparatus.51

As previously discussed, CaM is a promiscuous peptide binding protein. Typically the

interacting peptides all have common recognition motifs but on whole they have little sequence

similarity.52

Structurally, the peptide bound CaM structures are generally compact with either an

anti-parallel or parallel orientation. Similar biophysical studies with binding peptide present will

certainly enhance our knowledge of the CaM system. These studies could address whether some

bound conformations lead to a stabilization of the protein which reduces the fluctuations and if

fluctuations do still occur does the peptide bound state prevent inter-domain misfolding?

50

A study in calmodulin would not be complete without investigating the apo-state. Apo-CaM in

general is less thermo-stable, more compact, and less flexible than holo-CaM.53

Of particular

interest to our studies is the fact that the hydrophobic residues in apo-CaM pack together

between α-helical segments in each globular domain forming a hydrophobic core in the interior

of the structure, whereas in holo-CaM, the hydrophobic residues face outwards, adjacent and

perpendicular to the central helix, forming a hydrophobic core accessible from the outside. In

essence, the hydrophobic cores of the apo- and holo- state are structurally distinct from each

other. With the significant structural differences, it is not surprising that apo-CaM binds target

proteins differently making use of other motifs.53

In general, each CaM state has affinities to

different proteins and therefore each state performs different functions.52

All of these

characteristics make apo-CaM an equally important system to investigate.

Figure 37 A. X-ray structure of: Apo-calmodulin (PDB file 1CFD)54

B. Holo-Calmodulin (PDB

file 1CLL)55

.

An important parameter in our studies is the population of both states. Obtaining the populations

will allow us to define kfolding and kunfolding rates from the global kex. As mentioned, populations

can be obtained from CPMG data for exchange processes in the slow exchange regime, however

since the process of interest is in the fast regime, alternative approaches must be used. Currently

we are working on defining the populations through use of the near-UV CD data and the

51

diffusion data. In particular, the sigmoid-like shapes of Figures 15A and 17 are of importance

since we observe the initial state shifting in equilibrium to the intermediate state through a

transition. With the appropriate analysis, this data should allow us to define the populations of

each state at a given temperature.

52

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55

Appendix A-Full-Length CaM Dispersion Curves by Residue

Data were fit to a one phase decay.

56

Appendix B-TR1C CaM Dispersion Curves by Residue


57

Appendix C-TR2C CaM Dispersion Curves by Residue


protein folding and dynamics of calmodulin via f-nmr · calmodulin (cam) is a ubiquitous calcium...

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