biomolecular vibrational spectroscopy, pt. 2: applications ... · interaction between protein and...
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BioMolecular Vibrational Spectroscopy, Pt. 2:
Applications of Infrared, Raman, VCD Spectra
Lectures for Warwick CD Workshop, Dec. 2011
Tim Keiderling
University of Illinois
at Chicago
tak@uic.edu
Tentative Schedule — can vary with interests
Part I: • Vibrational Spectra Theory and Practice • IR - Raman Instrumentation; techniques
Part II: • Application Examples
– Peptide bases, secondary structure types results – Avoiding aromatics – Protein differences
• Isotopes give new dimension, site specific • Dynamics and ultra fast • Imaging (no time)
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 obscured
by 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
Wavenumbers (cm-1
)
1450150015501600165017001750
Ab
so
rban
ce
0
1
2
3 helix
-structure
randomcoil
IR absorbance spectra of selected model peptides
I II
IR absorbance spectra of selected model polypeptides
(LKKL)n
helix
(LK)n
sheet
polyK
Coil (31)
H2O solution D2O solution
L=Leu K=Lys
Proteins—shift the emphasis
Multiple structural types
IR bands overlap
Deconvolve - “resolution enhance”—derivatives, FSD
Band fitting - subjective
Bandshape (Factor or Principle Component) analysis
Extra insight—
Polarization - surface sensitive experiments (ATR)
Resonance Raman – chromophore emphasis
Imaging – biological tissue, distribution
Time Domain – reactions, folding, photolysis
Circular Dichroism—chirality
Myoglobin
Hemoglobin
Cytochrome C
Citrate Synthase
-3
-2
-1
0
1
1800 1700 1600 1500 1400
Wavenumber (cm-1)
Superoxide
Dismutase
-2
-1
0
1
1800 1700 1600 1500 1400
Wavenumber (cm-1)
Lectin
Concanavalin-A
‘No’ a-helix, high -sheet High a-helix, ‘no’ -sheet
Example Protein/H2O IR spectra:
A A
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’
FTIR
Inc. D2O
Difference IR
DFTIR
IRx-IRH2O
Amide I relatively small change, amide II 100 cm-1 shift, amide III more
ATR Polarization Measurements
E
IR beam multiply reflects inside crystal -- penetrates surface
keeps polarizations: Eperp in surface, Epara partially out
Sample coated
on crystal
Crystal-
Typ: Ge, ZnSe
High index for refl.
In from
FTIR
out
A ‘Few’-bounces ATR - Types available
• Often diamond over ZnSe
– Provides very hard surface
– Can apply pressure with rod above
• 1-3 bounces, signal can be small – solution difficult – Good for membranes, solids
– Easy to clean
• 9-bounce dip to hold liquid
• Design to fit your FTIR
• Several manufacturers
Structure of Bovine -lactoglobulin
Milk Protein Sequence predict: alpha helical Undergo beta to alpha transition with surfactants, lipids
Interaction between Protein and Lipid Vesicles
DMPG 14:0
-sheets a-helices
11
0 1 2 3 4 5
0.1
0.2
0.3
0.4
0.5
BpH 6.8
-Sheet
a-Helix
Unordered
Fra
ctio
nal
sec
ondar
y s
truct
ure
DMPG / mM
Secondary structure: Binding DMPG at pH6.8, causes BLG conformational change. The
a–helix formed with loss of -sheet.
2nd struct. analyses
BLG Binding to DMPG at pH 6.8: CD result
--sheet to a–helix transition, dependence on DMPG
12
X.Zhang,TAK Biochemistry 2007
Orientation of BLG into lipid membrane: -Polarized ATR-FTIR spectra of DMPG-bound BLG
3000 2900 1800 1700 1600 1500 1400 1300
1654 1637
2960
2912
2848
1731
1745 16
5416
37
1467
1343 13
2813
05 1255
1229
1280Dichroic spectrum, pH 6.8
Dichroic spectrum, pH 4.6
900 polarized spectrum
00 polarized spectrum
Wavelength / cm-1
helix sheet
CH2 str
CH2 scis
amide I
13 X.Zhang,TAK Biochemistry 2007
Time-resolved Spectroscopy Techniques
Stopped-flow CD and Fluorescence
Stopped-flow FTIR
P C
Protein
RenaturantDenaturant
Looking at the mechanism of the transition from initial to final state
Wavenumber (cm-1)
15501600165017001750
log
(S
i/S
f)
-0.03
-0.02
-0.01
0.00
0.01
0.02
1660 cm-1
loss of random coil
1630 cm-1
gain of sheet
time (s)
0 5 10 15 20
Peak In
ten
sit
y
-0.03
-0.02
-0.01
0.00
0.01
1632 cm-1
(sheet)
k = 0.156 s-1
1660 cm-1
(random coil)
k = 0.342 s-1
Refolding of Ribonuclease A by FTIR
Slow/Rapid scan FTIR - Inverse T-jump: Refolding initiated by
injecting Ribo A stored in syringe at 80 °C into IR cell at 25 °C
Sheet refolding 2x slower
than loss of coil
One single beam spectrum (IF scan) is collected for each time point.
Time res. = 50 ms. IR resolves increase coil & decrease sheet fold.
Difference IR
Heinz Fabian et al.
Concanavalin A pH, TFE and MeOH
native
TFE
MeOH normalizes -sheet ECD,
FTIR indicates aggregated
TFE induces helix Xu&Keiderling, Biochem, 2005
Stopped-flow FTIR of Con A Unfolding with TFE (1:1)
Fast Transition
(Principle Component Analysis) (Difference Spectra)
1630 cm-1
1654 cm-1
Qi Xu and TAK, Proteins
Wavenumber (cm-1)
1600165017001750
Absorb
an
ce
0.0
0.5
1.0
A
x 1
05
-10
-5
0
5
10
VCD
IR
(a)
Wavenubmer (cm-1)
1600165017001750
Ab
so
rba
nce
0.0
0.5
1.0
A x
10
5
-4
-2
0
2
IR
VCD
(b)
Poly Lysine in D2O – Amide I’–Secondary structure VCD
High pH – helix High pH, heating – sheet Neutral pH - coil
Wavenumber (cm-1)
1600165017001750
Absorb
ance
0.0
0.5
1.0
A
x 1
05
-15
-10
-5
0
5
IR
VCD
(c)
Wavenumbers (cm-1
)
140016001800
Ab
so
rba
nc
e
0
1
2
3
4
Wavenumbers (cm-1
)
140016001800
A
(A
.U.)
-100
0
100
200
300
400
500
a-helical
(Aib-Ala)6
Ala(AibAla)3
310
-helical
a-helical
310
-helical
Ala(AibAla)3
(Aib-Ala)6
VCD success example: 310-helix vs. a-helix
Relative shapes of multiple bands distinguish these similar helices
Aib2LeuAib5
(Met2Leu)6 a
310
mixed
i->i+3
i->i+4
Silva et al. Biopolymers 2002
Biphenyl bridged residues (Bip) CD and IR difficult to get structure
CD—all biphenyl Amide A shows H-bond form
Toniolo, co-workers JACS 2004
Biphenyl bridged residues (Bip) show inversion
VCD and IR spectra of Ac-(Bip)3-L-
Val-OMe (full lines) and Boc-L-Val-
(Bip)4-OtBu (dashed lines). Spectra
of Ac-(Bip)3-L-Val-OMe were
measured in 46/11 (v/v) CDCl3/TFE-
OH and Boc-L-Val-(Bip)4-OtBu in
CDCl3 solution, pathlength 500 mm
and concentration of 9.5 and 8.6 g/L,
respectively.
Ac-(Bip)3-L-Val-OMe (_________)
left-handed
Boc-L-Val-(Bip)4-OtBu (-------)
right-handed (310-helix)
Toniolo, co-workers JACS 2004
Vibrational spectrum separates
aromatic and amide transitions
Tiffany and Krimm in 1968 noted similarity of Proline II
and poly-lysine ECD and suggested “extended coil”
Problem -- CD has local sensitivity to chiral site
--IR not very discriminating
Nature of the peptide random coil form
Dukor and Keiderling 1991 with ECD, VCD, and IR showed
Pron oligomers to have characteristic random coil spectra
Suggests -- local order, left-handed turn character
-- no long range order in random coil form
Same spectral shape found in denatured proteins, short
oligopeptides, and transient forms
Dukor, Keiderling - Biopoly 1991
Relationship to “random coil” - compare Pron and Glun
IR ~ same, VCD - same shape, half size -- partially ordered
in CDCl
in TFE (Aib-Ala) 4
Wavenumber [cm -1
]
1500 1600 1700
Aib 5 -Leu-Aib 2
(Met 2 -Leu) 8
310-helix vs. a-helix: comparison of Aib, Ala and Aib-
Ala alternating sequences.
(Kubelka,Silva, Keiderling JACS 2002)
Simulation: a-helix
Experiment: Simulation: 310-helix
Wavenumber [cm-1
]
150016001700
/a
mid
e
Ac-(Aib)8-NH
2
Ac-(Aib-Ala)4-NH
2
Ac-(Ala)8-NH
2
Wavenumber [cm-1
]
150016001700
/a
mid
e
Ac-(Aib-Ala)3-NH
2
Ac-(Ala)6-NH
2
Simulation of Helix IR and VCD Really Works!
13C Isotopic Labeling
Change 12C to 13C on amide C=O
shift amide I down by ~40 cm-1 (isotopic shift) (13C=18O even more)
Isotopic advantage: site-specific
(specific, local Amide I vibrational coupling)
Amide I (1700-1600 cm-1)
coupling structure
IR limitation: average secondary structure
(delocalization of Amide I vibrational coupling)
Isotopes can break that - give specific sites
Basic principle of spectra-structure relationships:
Wavenumber [cm-1
]
1550160016501700
An
orm
(x 1
0)
0
4
8
12Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
165017001750
x 1
0-3
)
2
4
165017001750
Wavenumber [cm-1
]
1550160016501700
Unalbeled
N-terminus
C-terminus
Middle (N)
Middle (C)
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
Simulated & experimental amide I IR : Ala20 - 13C labels
a-helix ProII-like
Low T High T
Silva, Kubleka, et al. PNAS 2000
Simul.
Exper.
165017001750
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
165017001750
x 1
0)
-8
-6
-4
-2
0
2
Wavenumber [cm-1
]
1550160016501700
A
no
rm (
x 1
05)
-8
-4
0
4
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
Wavenumber [cm-1
]
1550160016501700
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
Unlabeled
N-terminus
C-terminus
Middle (N)
Middle (C)
a-helix ProII-like
Low T High T
Simulated & experimental VCD : Ala20 - 13C labels
Silva, Kubleka, et al. PNAS 2000
Temperature [oC]
10 20 30 40 50 60
Fre
quency [cm
-1]
1643
1645
1647
1649
1651
1653C-terminus
N-terminus
Unlabeled
Temperature [oC]
10 20 30 40 50 60
Unlabeled
Middle (C)
Middle (N)
a b
Frequency shift of 12C amide I’ VCD band minimum with temperature: a) terminal, b) middle labeled. Unlabeled added for comparison. Termini “melt” at lower temperatures
Silva, Kubleka, et al. PNAS 2000
Unstable termini – identify location with isotope
Center Termini
Relative isotope position-- experiment and theory
Two sequential labels have higher IR freq. due to coupling (intensity in high n mode), VCD : sequential (2LT) - same sign 12C and 13C, but opposite sign if separated (2L1S)
* since exp. in D2O a (-)band develops the amide I, but not modeled without solvent
IR VCD
*
13C
13C
13C
13C
Huang, et al. JACS 2004
. . .AKAAAAK. . .
. . .AKAAAAK. . .
. . .AKAAAAK. . .
. . .AKAAAAK. . .
Coupling is source of variation
N C
Wavenumber [cm-1]
Simulations for antiparallel -sheet - strands offset by one residue
Wavenumber [cm-1
]
1600164016801720
Absorb
ance
*
*
*
Kubelka, Huang unpublished, 2004 - best fit!
Simulation
L34
G38
V39
Hairpin – simplest -sheet model Monomeric model - anti-parallel interaction Interpretive challenge — heterogeneous Turn very different from strands Strands are non-uniformly twisted Ends are frayed Stabilize hairpin – two methods Turn residues that lock in limited conformation
--DPro-Gly (isotope interference, Hilario, et al. JACS 2003), --Aib-Gly (Setnicka, et al. JACS 2005, Huang, et al. JACS 2007)
Chain residues interact (hydrophobic, charges) --Trp-Zip models (Cochran, PNAS 2001, many followups), --aromatic coupling (Waters, others)
heating from 5 to 85°C, step 5°C
TrpZipper
AsnNH
NH
NH
NH
NH
NH3
+
Ser
O Trp
NH
NH
NH
NH
NH
NH
Lys O O
OO
O
O
O
O
OO
Thr
Trp
Glu
Trp
Thr
Trp
Lys
NH2
O
-O
Stable hairpin – cross-strand hydrophobic interaction
Trp-Trp interactions stabilize hairpin - Cochran et al. PNAS 2000
Trp interfere in CD – provides no secondary structure information Effectively tertiary
5 C
85 C
ECD intensity change vs temperature at different pH
Temperature (K)
280 300 320 340 360
[ ] x
10
- 5 (d
eg
cm
2 d
mo
l-1
)
-8
-4
0
4
8
12
pH=6.94 at 226.8nm
pH=2.38 at 227nm
pH=6.94 at 212.8nm
pH=2.38 at 212.6nm
c
Reversable, 215, 230 nm same transition
Secondary Structure Change with
Temperature - Monitor with IR
Thermal transition from -hairpin
(low temp, 1625-30 cm-1 max.) to
disordered (high temp, 1645-50
cm-1 max.)
TZ2C intensity change at acidic pH
Temperature (K)
260 280 300 320 340 360 380A
bs
orb
an
ce .20
.25
.30
.35
.40
.45
.50
.55
c
intensity change at 1632.2cm-1
intensity change at 1652.5cm-1
Tm=321.1KTm=332.8K ____
IR intensity change at 1632 cm-1 (-
sheet, black), 1652 cm-1, (disordered,
red), fit to a two state model - transition
temperatures of 321 K and 333 K
not really two-state!! Huang, Wu, et al. JPhysChemB 2009
13C=O isotopic labeling of TZ2C
Simulation
Experiment H3N+
HN
NH
HN
NH
H2N
HN
NH
HN
NH
O O
HN
O
O
OO
Ala
Lys
Ala
Ala
Glu
Lys
HN
Trp
Trp
Trp
Trp
O
O
O
O O
NH
O
Asn
H3N+
HN
NH
HN
NH
H2N
HN
NH
HN
NH
O O
HN
O
O
OO
Ala
Lys
Ala
Ala
Glu
Lys
HN
Trp
Trp
Trp
Trp
O
O
O
O O
NH
O
Asn
H3N+
HN
NH
HN
NH
H2N
HN
NH
HN
NH
O O
HN
O
O
OO
Ala
Lys
Ala
Ala
Glu
Lys
HN
Trp
Trp
Trp
Trp
O
O
O
O O
NH
O
Asn
Cross-strand vibrational coupling is
position sensitive
A1A10
A3A10
A3K8
13C
13C
Simulations predict 13C=O coupling
DFT-level FF, NMR structure
Huang, Wu et al. JPhysChem B 2009
Summary – equilibrium variation Tm values differ for 12C and 13C modes Also differ for frequency shift and intensity change Inconsistent with 2-state model for unfolding Fits earlier conclusions, folding multistate ensemble
Dynamics might give mechanistic insight
Trpzip-based beta hairpin temperature jump IR studies enhanced by site-specific
isotope labeling
with Karin Hauser group
University of Konstanz
Previously included: Carsten Kretjschi1, Rong Huang2, Ling Wu2, Tim Keiderling2
1Institute of Biophysics, University of Frankfurt,
2Department of Chemistry, University of Illinois at Chicago
Or: why I am in Konstanz as Humbolt Fellow
5-8 µm
Nd:YAG Laser
1064 nm, 700 mJ, 10 ns
IR Laser Module
Sample
IR
Detector
1906 nm
Raman-Shifter
Tunable semiconductor lasers as IR source:
• amide I single wavelength transients
• spectral range: 2300 -1200 cm-1
• modes every 2 - 4 cm-1
• cw power up to 1 mW
• high S/R
Laser-Induced Temperature Jump &
Single Wavelength Detection
• Conformational relaxation dynamics in the ns-µs time range
• Excitation of water overtone/combination vibration at 1906 nm
40 °C; t = 1.1 ± 0.1 µs
34 °C; t = 1.8 ± 0.1 µs
27 °C; t = 2.4 ± 0.1 µs
21 °C; t = 3.5 ± 0.2 µs
15 °C; t = 5.8 ± 0.5 µs
52 °C; t = 0.6 ± 0.1 µs
46 °C; t = 0.8 ± 0.1 µs
Unfolding Dynamics of TZ2C
• relaxation kinetics at
initial temperatures
• single exponential fit:
A(t) = B exp(-t/t)
1700 1690 1680 1670 1660 1650 1640 1630 1620 1610 1600
-0.20
-0.15
-0.10
-0.05
0.00
0.05
85°C
5°C
5°C
85°C
A /
OD
wavenumber / cm-1
-hairpin disordered
24 °C; t = 2.9 ± 0.2 µs
30 °C; t = 2.2 ± 0.1 µs
36 °C; t = 1.7 ± 0.1 µs
42 °C; t = 1.3 ± 0.1 µs
11 °C; t = 5.6 ± 1.2 µs
18 °C; t = 4.5 ± 0.4 µs
-loss faster Hauser, et al. JACS 2008
T-variation
Arrhenius
b
c
A1A10
A3A10 A3K8
Dynamics Labeled TZ2C 13C=O dynamics picks out
character of region
Blue—-strand Red – disordered Green – 13C=O relaxation
Hauser, et al. JACS 2008
>C=O H-N<
Two β-forms of P-L-GA and their influence on FT-IR – follows earlier work with insulin fibril formation
>C=O H-N<
H-O-C=O
1680 1660 1640 1620 1600 1680 1660 1640 1620 1600
18 cm-1 A
bw
sorb
ance
, a. u
.
Wavenumber, cm-1
1
0
50
Aggregating, self-assembled peptides (proteins)
Collaboration with Wojciech Dzwolak, Univ. Warsaw
1 form ……. 2 form ___ bifurcated H-bond
normal structure
Low pH, incubate, heat
52
TEM
SEM
Twisted fibril formation with pure enantiomers
L D L+D Fulara et al. J Phys Chem B 2011
1800 1750 1700 1650 1600 1550 1800 1750 1700 1650 1600 1550
2
1
-1
0
VCD Amplification of long range order
2L+D
L
53
2
0
2
4
1800 1750 1700 1650 1600 1550 1750 1700 1650 1600 1550
3L +D 2L +D 1.5L+D 1.25L+D
Chiral titration of L-Glu with D-Glu – only small change in IR
Fulara et al. J Phys Chem B 2011
-COOH
Acknowledgments
UIC
Helices Gangani Silva (now U.Cincinnati)
Rong Huang (now pd. Cincinnati)
Hairpins and sheets Ling Wu, (now pd Mich.State)
Heng Chi, Ahmed Lakhani
Theory
Jan Kubelka, (now U.Wyoming)
Joohyun Kim, (now LSU comp.)
Petr Bour, Acad. Sci. Prague
Anjan Roy
Vesicle-Protein - Xiuqi Zhang,
Ning Ge, Ge Zhang, Weiying Zhu
NMR Dan McElheny – UIC
T-jump IR Prof. Karin Hauser
Carsten Krejtschi
Frankfurt & Konstanz
Aib-Gly Hairpins Prof. Robert Hammer
Marcus Etienne, LSU
Aggregating peptides Wojciech Dzwolak - Warsaw
Funding: NSF, Guggenheim and Humboldt Foundation 54
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