BioMolecularBioMolecular Optical Spectroscopy: Optical Spectroscopy: Part 2: Infrared and Raman Part 2: Infrared and Raman

Vibrational Spectra Vibrational Spectra –– BioBio--applicationapplication

Special Special Lectures for Lectures for ChemChem 344344Fall, 2007Fall, 2007

Tim Tim KeiderlingKeiderlingUniversity of Illinois at ChicagoUniversity of Illinois at Chicago

tak@uic.edutak@uic.edu

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Vibrational Spectroscopy Vibrational Spectroscopy -- Biological ApplicationsBiological Applications

There are many purposes for adapting IR or Raman vibrational spectroscopies to the biochemical,

biophysical and bioanalytical laboratory• Prime role has been for determination of structure. We will

focus early on secondary structure of peptides and proteins, but there are more – especially DNA and lipids

• Also used for following processes, such as enzyme-substrate interactions, protein folding, DNA unwinding

• More recently for quality control, in pharma and biotech

• New applications in imaging now developing, here sensitivity and discrimination among all tissue/cell components are vital

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Motivation: Structural BiologyMotivation: Structural Biology

• Define Protein/Peptide structure various environments -- What

• Determine folding mechanisms— How did it get that structure

• Use these tools to monitor structural change during biological process--assay

• Relate peptide folding to protein mechanisms—TAK group

GOALS (Protein/peptide):GOALS (Protein/peptide):

General Principle:Establishing the molecular structure of complex biological systems can lead to understanding and eventually control of function (Pharma application)

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Amino AcidsVariation in the side-chains leads to stability in the fold and makes protein function possible. This key element of protein is typically not studied with vibrational spectra, exceptions are aromatics, disulfides – with Raman and carboxylates, charged groups – with IR (especially difference spectra, and PO2

- in DNA).

The backbone (amides) are the usual focus of IR and Raman, and of far-UV CD.

Fluorescence and near-UV CD do focus on aromatic residues and prosthetic/ligand groups T

Peptide Primary and Secondary StructureProteins are polymers of amino acids (20 natural)—Peptides more

Pro-Ala-Val-His-Ala-Ser-Leu-Asp-Lys-Phe-Leu-Ala-Ser-Val-Ser-Thr-Val-Leu

Secondary Structure —stereochemical relation of residues in chain—near neighbor repeat

Helix –end-on

Helix –side-on

Primary structure—sequence of residues (amino acids) in chain—charged, polar, hydrophobic, steric constraints

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Tertiary and Quaternary Structure

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Tertiary – fold/packing of secondary structure segments in one chain – hydrophobic, S—S, salt bridge, ligand, etc.

Quaternary – interaction of subunits or separate components (not covalently bound)

Mb Hb

[4 subunits]

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DNA bases and ribose phosphate chain

Structure variation leads to characteristic spectra—especially duplex formation

Watson-CrickBase pairing

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DNA duplex conformations

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t-RNA folding –single strand with hairpins forming duplex segments

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Biological Structures: Biological Structures: PhosphoPhospho--lipidslipids

Polar head – can be other functionalities-can attach sugars

Hydrophobic tail – can be single, - can be unsaturated

Ester and Phosphate links strong IR bands

Not much UV or CD

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Biological Structures: LipidsBiological Structures: Lipids

Lipid Bilayer

Lipid is amphipathic Liposome

Micelle

Polar head

Hydrophobic tail

Note: lends itself to orientation andpolarization studies

Self-assembly is a major structural aspect of lipids, impact proteins

Single layers also possible, on air-water interface, also lung alveoli

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Biological Structures: LipidsBiological Structures: Lipids

Interactions of membrane proteins with the lipid bilayer

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Biological Structures: CarbohydratesBiological Structures: Carbohydrates

Sugar structures have strong repeat similarity making discrimination with spectral techniques difficult. Typically they have little UV absorbance or CD. Ring conformation, glycosidic link and chirality offer useful elements to probe –show up in IR, Raman (esp. ROA).

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Conventional Structural TechniquesConventional Structural Techniques

Atomic resolution—Great advantages - at a cost!

X-RAY Crystallography – great precisionDisadvantages - must have a crystal/solid state

Nuclear Magnetic Resonance (NMR)-solution!Disadvantages - smaller proteins, high conc., large sample

Computational Methods - quick, easy for small systemDisadvantages - reliability in question, scaling up is problematic

Incredibly valuable, but can not solve all problems

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Level of structure determination needed depends on the problem

Atomic resolution Cα chain

Secondary structure Segment fold (tertiary)

Complex but often repeats (2nd struct.)

Protein Structure

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Low Resolution Conformational TechniquesLow Resolution Conformational TechniquesOPTICAL SPECTROSCOPY— topic of this course

UV absorption and fluorescence, --electronic transitions

IR absorption, Raman scattering, -- vibrational transitions

Circular Dichroism – polarization of transitions (due to chirality)

Advantages: all phases - gas, solution, crystal, film,

conformationally sensitive to secondary structure and sometimes to tertiary structure

Time scale on order of nuclear motion- Can follow Dynamics

Disadvantages: not site specific- averages

For amide bond as chromophore, UV absorbance broad and little fluorescence—CD or IR/Raman offer best alternatives

With nucleic acids, bases have characteristic but broad UV bandsCarbohydrates and lipids have little UV of use

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Structural BiologyStructural Biology

Optical SpectroscopyOptical Spectroscopy isis limitedlimited for determining for determining structurestructure butbut often fits importantoften fits important QUESTIONSQUESTIONS

• often need to know just the conformation

• structural determination of fold family may suffice, generally not after atomic structure

• In BioTech processes one must monitor effect of mutation and environmental changes

need to get this information rapidlyand in a cost effective manner

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Biologically Relevant Structures and Their Biologically Relevant Structures and Their SpectraSpectra

A. Peptides

B. Proteins

C. Nucleic Acids

D. Carbohydrates

E. Lipids

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PeptidePeptide--Protein IRProtein IR--Raman examplesRaman examples

• PEPTIDE / PROTEIN spectroscopy

– basically reflects amide bond linkage and its interactions,

– leads to secondary structure interpretation

• Side chains

– contribute in few specialized roles, in IR

– But in Raman can be important, especially aromatics, S-S

• Amino acids are rarely subject to IR/Raman study

• Ligands and prosthetic groups are often target of differential IR and Raman

• Special methods—polarization, imaging and time dependence now coming into importance

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Chain conformation depends on Chain conformation depends on φφ, , ψψ anglesangles

If (φ,ψ) repeat, they determine secondary structureIR-Raman analyze polymer chain via amide coupling T

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Characteristic Amide Vibrations

I - Most useful;IR intense, less interference (by solvent, other modes,etc)Less mix (with other modes)

II - IR intense

III - Raman Intense

A – often obscuredby solvent

IV – VII – difficult to detect, discriminate

~3300 cm-1

~1650 cm-1

1500-50 cm-1

1300-1250 cm-1

700 cm-1

mix

Also Raman

Not Raman, unless RR

Weak IR Multiple bands

αα--helix helix -- common peptide secondary structurecommon peptide secondary structure

(i i+4)

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ββ--sheet crosssheet cross--strand Hstrand H--bondingbonding

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AntiAnti--parallel parallel ββ--sheet (extended strands)sheet (extended strands)

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Model polypeptideModel polypeptide IR absorbanceIR absorbance spectra spectra -- Amide Amide II and and IIII

Wavenumbers (cm-1)

1450150015501600165017001750

Abs

orba

nce

0

1

2

3 helix

β-structure

randomcoil

III

(weak IR but strong in Raman)

(Not in Raman)

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Amide I ModesAmide I Modes --POLYPEPTIDESPOLYPEPTIDES• All regular infinite polymers will have three dipole allowed modes

(along x, y, z) due to a combination of all the local C=O stretching (amide I) motions

• α-helix shows two overlapped bands (intense z-polarized parallel helix& weak x,y - perpendicular) protein ~1655 cm-1, solvated peptide (D2O)~1635 cm-1

• β-sheet has two or three bands, large split between x,y (lower νperpendicular one intense), z is weak extended anti-parallel: 1620-15 (s) & 1690-80 (w) cm-1

• random coil is not regular, not polarized ~1640-45 cm-1, broad

• RAMAN frequencies different due to different components of polymer band having intensity

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Vibrational Frequencies For PolypeptidesVibrational Frequencies For Polypeptides

• Theory--Normal mode analysis of ideal polypeptides. Assumptions:

– ideal conformations

– infinite length

– force constants independent of side chains

• Empirical—IR/Raman spectra of homopolymers in various conformations

Interpretive sources:

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IR absorbance spectra of selected model polypeptidesIR absorbance spectra of selected model polypeptides

TWavenumbers (cm-1)

1450150015501600165017001750

Abs

orba

nce

0

1

2

3 helix

β-structure

randomcoil

III

H2O solution

(LKKL)nhelix

(LK)nsheet

polyKCoil (31)

D2O solution

L=LeuK=Lys

Raman spectra of polyRaman spectra of poly--LL--lysine in three lysine in three different conformationsdifferent conformations

I II III

Note: β-sheet amide I, opposite band is intense (high ν)Amide III large frequency shift, mixing with CαH

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IR Linear Dichroism - α-helical polypeptideAmide I || helix axis, Amide II ⊥

%T

⊥ - - - - - - ⎥⎪ _______

Amide IIAmide I

Oriented film of poly-γ-dansyl-L-glutamate – from Tsuboi J. Pol. Sci. 1962T

IR Polarization indicates structureIR Polarization indicates structure

α-helical naturalextended proteinsI – parallel polarizedII - perpendicular

β-sheet extended protein (silk)I, A – perpendicular II - parallel polarized

wool quill

Parallel - - - - Perpendicular_______

Amide I

Amide IIα

β Amide AI II

Solid phase samples, oriented fibersT

CONFORMATION DEPENDENCECONFORMATION DEPENDENCE

• Conformational dependence (φ, ψ) of frequency is one of several features that effect the amide group frequency.

• HYDROGEN BONDING

– H-bonding will change the strength of hydrogen bonds that occur in ordered secondary structures. Stretching vibrations to lower energies (Amide I, Amide A) and bending vibrations to higher energies (Amide II)

• TRANSITION DIPOLE COUPLING

– Each vibrating group feels the transition dipole of neighboring groups. In ordered polymers, this effect becomes significant

• OTHER EFFECTS ON FREQUENCY

– Solvent, dielectric (pH, salt), residue (charged, aromatic)

Bottom line—take assignments with grain of salt!T

Conformational Marker Bands: IR and RAMANConformational Marker Bands: IR and RAMAN

Structure Amide I (H2O) Amide I’ (D2O) Amide III Skeletal C-C β-Sheet (extended) 1640-1620 cm-1 1635-1615 cm-1 1685-1675 ; 1665-1680 1680-1670 1240-1225 1010-1000 Aggregate* 1695 1690 1615 1610 α-Helix 1658 ; 1660-1645 1655 1310-1260 950-885 310-Helix 1660 1638 Turns 1675-1660 1670-1660 ‘Random’ (unordered) 1650 ; 1670-1660 1645 1260-1240 960-950 *Seen in denatured forms of proteins.

Color code: Blue—Raman, black--IRT

Amide A IR, non-polar solvent

octapeptide

tetrapeptide

Free N-H

H-bonded N-H

Increase of intensity at ~3300 cm-1 hydrogen bond formation consistent with formatin of 310-helix

(Aib)n -(L-Leu)-Aib2 oligomers (blocked)Yasui et al. JACS 1986 T

Amide I - II study of (αMe)Valn oligomers in CDCl3

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octapept.

tripept.

Amide I - II IR

I II

Increased length closes the amide I – II gap, and bands sharpen – implies more uniform 310 helix, nature of helix confirmed by VCD

Yoder et al. JACS 1997

Ala-rich α-helical peptide in aqueous solution

Ac-(AAKAA)n-GY-NH2 – α-helicalNote-TFA interference in amide I

H2O—high conc. short path

TFAII

ID2O—only amide I’-low ν, solvated

TFA n = 4

n = 3

n = 2

n = 1

helixsharper

coilbroader

Conformations confirm with CD

Yoder et al., Biochemistry 1997 T

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Helix formation of biphenyl bridged AA peptide - length

Boc-L-Val-(Bip)4OtBu Ac-(Bip)3-L-Val-Ome

I IIIR

L-Val-(Bip)n(Bip)n-L-Val VCD

BIP

Amide A IR

High ν, no info.

Poly-Proline forms, I-right-handed & II-left-handedII I II I

Left-handed PLPII has C=O groups pointing out to solvent—favored for coil, disorder form

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Proline oligomers stabilize ProII or 31-helix-tertiary amides make lower frequency

Multiple componentsdifferent conformations

Equivalent to polymerIR Trace growth of helical form, with length – stabilitySharper, well-formed band implies coherent structure

Dukor&Keiderling, Biopolymers 1981 T

Mutarotation of ProI ProII study with IRI

mix

III

IR shows change from I II is not simple, 1650 cm-1 band grows in and decays with time

Dukor&Keiderling, BiospectroscopyT

Thermal analysis of unfolding

Simple methods--frequency variation of a peak--intensity variation at a frequency

Bandshape methods -- Factor analysis used--determine most common components--determine loadings of each component in each spectrum--analyze variation of loadings vs. Temperature--Varimax rotation optimize projection onto specific form

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Wavenumbers (cm-1)

15751600162516501675170017251750

Abs

orba

nce

0.0

0.1

0.2

0.3

0.4

0.5

Temperature dependent absorbance Temperature dependent absorbance -- Ala rich 17mer Ala rich 17mer -- aqueousaqueous

TFA subtracted and rescaled FTIR spectra of the thermal unfolding of the unlabeled 17mer peptide at temperatures from 5o C to 45o C see shift up in wavenumber, helix to coil, unusually low helix frequency Example of problem of solvent shift frequency (linear peptide exposed)

5o C

45o C

Ac-YAAKAAAAKAAAAKAAH-NH2

Factor analysis - difference FTIR - Ac-(AAKAA)4-GY-NH2

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Second component rise and fall indicates intermediate between helix and coil. Consistent with unwinding from ends Yoder et al. Biochemistry, 1997

Transfer of FF, APT and AAT (e.g. Ala7 to Ala20)

Main chain residues

Middle residueN-terminus C-terminus

20-mer

7-mer: FF, APT, AAT calculated at BPW91/6-31G* levelKubelka, Bour, et al., ACS Symp. Ser.810, 2002

Method from Bour et al. J. Comp Chem. 1997

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Isotope labeling site specific structure

Method - Change 12C to 13C on amide C=OShift frequency down by ~40 cm-1 (out of amide I)Decouple from rest of the chain

IR can detect differences, but frequencies alone not reliableVCD can determine the type of secondary structure

With peptide synthesis, straightforward, affordable for AlaPrevious studies coupled Experiment and Theory - observational

1. α-helix thermal denaturation (Silva et al, PNAS 2000)2. β-sheet formation - aggregation (Kubelka & TAK, JACS 2001)3. α-helix—coupling of sites (Huang et al. JACS 2004)4. β-hairpin—cross-strand coupling (Setnicka, et al. JACS 2005)

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Alanine 20-mer 13C labeling scheme

Notation Label position Peptide sequence

unlabeled none Ac-AAAAKAAAAKAAAAKAAAAY-NH2

L1 N-terminus Ac-AAAAKAAAAKAAAAKAAAAY-NH2

L2 Middle (closer to N-terminus) Ac-AAAAKAAAAKAAAAKAAAAY-NH2

L3 Middle (closer to C-terminus) Ac-AAAAKAAAAKAAAAKAAAAY-NH2

L4 C-terminus Ac-AAAAKAAAAKAAAAKAAAAY-NH2

Label clusters of 4 Ala to enhance signal and keep coupling

Silva, Kubleka, et al. PNAS 2000T

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Wavenumber [cm-1]

1550160016501700

Ano

rm (x

10)

0

4

8

12UnlabeledN-terminusC-terminusMiddle (N)Middle (C)

165017001750

ε (x

10-3

)

2

4

165017001750

Wavenumber [cm-1]

1550160016501700

UnalbeledN-terminusC-terminusMiddle (N)Middle (C)

UnlabeledN-terminusC-terminusMiddle (N)Middle (C)

UnlabeledN-terminusC-terminusMiddle (N)Middle (C)

Simulated and experimental IR absorption – labeled Ala20Vary from N- to C terminal, 4 13C sequential, C-term unfolded, high T agreement

α-helix ProII-like

Low T High T

Silva, Kubleka, et al. PNAS 2000

Exp.

Sim.(theory)

(coil)(helix)

Transfer of property tensors ( Bour et al, J. Comp. Chem. 18, 646, 1997)

source “small” molecule:FF, APT, AAT from DFT: BPW91/6-31G**

target “LARGE” molecule empirical couple FF, APT, AAT

The small molecule “overlaps” residue type with all corresponding parts of the target structure - local interactions between fragments and in strand are included .

Parameters from the edge ends are transferred onto the edge corners.

Parameters from the inner strand ends are transferred onto the inner ends.

Parameters from inner center residue transfer onto the inner strand residues (bulk of the sheet amides).

β-sheet applications: Kubelka & Keiderling JACS 2001. T

13C isotopic labeling (on C=O) in K2(LA)6 peptide

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Abs

orba

nce

(nor

mal

ized

x10

)

2

4

ε/ amide (x10

-2)

4

8

K2(LA)6Ac-A12-NH-CH3

Ac-AAA*AA*A7-NH-CH3

2-strands 3-strands 5-strands

Experiment Simulation (increasing number of strands)

2

4K2L*A*(LA)5

2

4

6

Wavenumber [cm-1]

160016501700

2

4

160016501700

2

4

Wavenumber [cm-1]

160016501700160016501700

2-strands 3-strands 5-strands

5-strands3-strands2-strands

Ac-AA*A*A9-NH-CH3

K2LA*LA*(LA)4

(sequential)

(alternate)

(unlabeled)

Simulation--Kubelka & Keiderling JACS 2001.Original Experiment -- Mendelsohn, co-workers JACS 2000

Sensitivity jump

Classic β-sheetaggr.

Sensitivity to structure from multiple strands

Origin of the “anomalous” 13C intensity enhancementRelative phases of the most intense modes in 5-stranded β-sheet modelunlabeled

1625.2 cm-1

-3 0 3 -3 0 3

C=O stretch

-3 0 3 -3 0 3 -3 0 3

alternately labeled

* **

** *

****

-2 0 2 -2 0 2

C=O stretch

-2 0 2 -2 0 2 -2 0 2

1650.7 cm-1

same phase pattern

lowest (π, 0) mode involves 13C labeled amides12C amide C=O poorly couple with this phase

(π, 0) phase, predominantly center strandsInner strands decoupled from the outer strands

(0, π) vibration needs extended structure, at least two coupled strands (H-bonded, central C=O - lower frequency, higher dipole):

→ enhancement seen in four- and five-stranded β-sheets→ extended strands (no twist) support multiple stranded β-sheets

Kubelka & Keiderling JACS 2001. T

Cross-strand hairpin labeling pattern

small H- bonding ring large H-bonding ring

Setnicka et al. JACS 2005T

Two labeling patterns, distinct coupling across strands

Simulation Experiment

Simulation gets the 13C=O part right on, but 12C=O, amide I, width is poorly represented—due to fraying of the hairpin

Setnicka et al. JACS 2005T

Hairpin labeling works - Site-specific folding

IR spectra of labeled Gellman A peptide:IR spectra of labeled Gellman A peptide:heating from 5 (heating from 5 (violetviolet) to 85) to 85°°C (C (redred), step 5), step 5°°C C

Wavenumber, cm-1

1600165017000.0

0.2

0.4

0.6

A

labeled onlabeled on Val3 and Lys8Val3 and Lys8

NH

NH

NH

NH

NH

NH3+

Arg

O TyrNH

NH

NH

NH

NH

NH

Gln O O

OO

O

O

O

O

O O

Val

Glu

Val

Leu

Ile

Lys

OrnNH2

O

Lys

IR

Result, labeled amides couple cross-strand, but after unfolding, coupling is lost and intensity falls off dramatically

Setnicka et al. JACS 2005T

Intensity Intensity 1212C=O and C=O and 1313C=O vs. TemperatureC=O vs. Temperature

TSetnicka et al. JACS 2005

Note isotope substituted band is most sensitive to temperature, also has sequence specificity

Proteins—shift the emphasis

Multiple structural typesIR bands overlapDeconvolve - “resolution enhance”—derivatives, FSD

Band fitting - subjectiveBandshape (Factor or Principle Component) analysis

Extra insight—Polarization - surface sensitive experiments (ATR)Resonance Raman – chromophore emphasisImaging – biological tissue, distributionTime Domain – reactions, folding, photolysisCircular Dichroism—chirality

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Example Protein/HExample Protein/H22O IR spectra:O IR spectra:

High α-helix, ‘no’ β-sheet ‘No’ α-helix, high β-sheet

SuperoxideDismutase

-2

-1

0

1

1800 1700 1600 1500 1400

Wavenumber (cm-1)

Lectin

Concanavalin-AMyoglobin

Hemoglobin

Cytochrome C

Citrate Synthase-3

-2

-1

0

1

1800 1700 1600 1500 1400

Wavenumber (cm-1)

AA

T

Amide III Amide III –– weak but correlatedweak but correlated

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FTIR and DFTIR of Lysozyme in H2O-D2O Mixtures

0

.2

.4

.6

.8

1

1750 1700 1650 1600 1550 1500 1450 1400 1350

100% H2O 100% D2O

-.4

-.2

0

.2

.4

1750 1700 1650 1600 1550 1500 1450 1400 1350

I II II’

FTIRInc. D2O

Difference IRDFTIRIRx-IRH2O

TAmide I relatively small change, amide II 100 cm-1 shift, amide III more

FTIR labeling experiments: FTIR labeling experiments: calmodulincalmodulin in Din D22OO

unlabeled

15N

13C/15N

Note the change in frequencyof Amide I band

Not yet site-specific like peptides, but coming

--not very useful for IR

--amide I shift ~40 cm-1

13C=18O more (~70 cm-1) but incomplete label

I

II

III

II’ - HOD

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Manipulating Spectra to Gain InformationManipulating Spectra to Gain Information

• Spectral Subtraction

• Pattern Recognition Techniques

• Derivatives

• Fourier Self-Deconvolution

• Curve Fitting

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Spectral SubtractionSpectral Subtraction

• Remove Unwanted Bands from a Spectrum

– Such as Liquid Water, Water Vapor, Excipients

• Sample – Reference = Result

– Sample => Mixture Spectrum (red)

– Reference => Pure Spectrum (blue)

– (Glutamine + Water) – (Water) = Glutamine (green)

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ANALYSIS: 2nd derivativeANALYSIS: 2nd derivative

• For multiple overlapped components, like protein spectrum, useful for determining the number and frequencies of components – good for qualitative discrimination

• Minima in 2nd derivative correspond to maxima in positive absorption in the original spectrum (in principle also find inflection points)

• The height is proportional to the curvature – sharper features emphasized (H2O vapor, Tyr)

• Common use for quantitative secondary

structure determination theoretically

unsound (but it is used anyway)

• Opposite signed wings confuse separation

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ANALYSIS: 2nd derivativeANALYSIS: 2nd derivative

Absorbance / Wav enumber (cm-1) Y-Zoom CURSOR

-.04

-.02

0

.02

1700 1600 1500

IR absorbance

2nd derivative

Note-2nd derivatives are negative for peaks, some software flip them- structure in high ν region – vapor, same lower plus noise

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ANALYSIS: Fourier Self ANALYSIS: Fourier Self DeconvolutionDeconvolution

• GOAL:

– enhance the apparent resolution of overlapping peaks, i.e. to distinguish contributions to multicomponent bands which are unresolved

• FSD does NOT increase the instrument resolution

• The amount by which the band width can be reduced is limited by the resolution at which the spectrum was originally measured

• Band area stays constant (ideally)

• Peak position is retained

• Peak amplitude is changed

• Problem: assume all components behave the same, same natural width, uniform frequency for structure

T

T

Fourier Self deconvolved Amide I – Ribonuclease SBand fit result to Lorentzian shapes, assign, analyze

(Byler&Susi, Biopolymers 1986)

β

β

α

t

rc More sheet than helix, helix probably 2 types, turns not quantiative

2nd derivative and 2nd derivative and deconvolutiondeconvolution::get same number of bands, same positionget same number of bands, same position

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rbance / Wav enumber (cm-1) Y-Zoom CURS

0

.05

.1

.15

1700 1600 1500

Protein FTIR

Second derivative

FSD

Crystal Structure of Horse Cytochrome c (3CYT)A helical bundle structure in the native state, but can access several partially folded states

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Helices

C-terminusLoops

N-terminus

Cyt c thermal unfolding followed by Factor Analysis of Amide I’ IR

native MG A Acid denatured

Note: MG and native aggregate, differ in Tm, U state further unfolds

UN

T

Raman Spectroscopy of ProteinsRaman Spectroscopy of Proteins

• ADVANTAGES:• no water interference

• sidechain and disulfide analysis

• can do micron focus

• DISADVANTAGES:• Requires high concentration

• Fluorescence interference

• More expensive and more complicated than IR

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RAMAN Protein SpectraRAMAN Protein Spectra

9.0 x 108

a) human serum albumin

IR + IL

1299 1342

c) hen lysozyme

6.3 x 108

IR + IL

13451241 162.5 x 109

b) jack bean concanavalin A

IR + IL

T

800 1000 1200 1400 1600

wavenumber / cm-1 Barron et al.

Raman Protein Raman Protein AmideAmide Marker BandsMarker Bands

T

ProteinProteinRaman Raman

AromaticAromatic

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Comparison of DNA / RNAComparison of DNA / RNA

solidsolution

0

.1

.2

1800 1600 1400 1200 1000

0

.2

.4

Wavenumbers / cm-1

0

.02

1800 1600 1400 1200 1000

0

.02

Abso

rban

ce

Wavenumbers / cm-1

(a)

(b)

*

DNA

RNA

Ribose rings differ between RNA and DNA, this shows up in the 1070 – 1120 cm-1 band (sym PO2 plus ribose C-O). Bases similar

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DNA IR Marker BandsDNA IR Marker Bands

T

DNA Base IR

Differentiation of Cytidine (a,b) andmethyl derivativeswith FTIR

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FTIR spectroscopy of Nucleic AcidsFTIR spectroscopy of Nucleic Acids

H2O

D2O

I: in-plane base double-bond vibrations -sensitive to base pairingII: base-sugar bending motions, sensitive to variation in glycosidictorsion angleIII: phosphate group vibrationsIV: phosphate-sugar backbone vibrations, sensitive to sugar puckering

T

LIPIDSLIPIDS

Often studied with ATR spectroscopy, self-assemble

Hydrocarbon, CH2 wag region

More later with polarization examples

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LIPIDS IR Marker BandsLIPIDS IR Marker Bands

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