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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
[email protected]@uic.edu
T
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
T
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)
T
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
T
Tertiary and Quaternary Structure
T
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]
T
DNA bases and ribose phosphate chain
Structure variation leads to characteristic spectra—especially duplex formation
Watson-CrickBase pairing
T
DNA duplex conformations
T
t-RNA folding –single strand with hairpins forming duplex segments
T
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
T
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
T
Biological Structures: LipidsBiological Structures: Lipids
Interactions of membrane proteins with the lipid bilayer
T
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).
T
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
T
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
T
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
T
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
T
Biologically Relevant Structures and Their Biologically Relevant Structures and Their SpectraSpectra
A. Peptides
B. Proteins
C. Nucleic Acids
D. Carbohydrates
E. Lipids
T
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
T
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
T
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)
T
ββ--sheet crosssheet cross--strand Hstrand H--bondingbonding
T
AntiAnti--parallel parallel ββ--sheet (extended strands)sheet (extended strands)
T
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)
T
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
T
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:
T
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
T
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
T
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
T
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
T
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
T
T
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
T
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
T
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)
T
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
T
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
T
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
T
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
T
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
T
Manipulating Spectra to Gain InformationManipulating Spectra to Gain Information
• Spectral Subtraction
• Pattern Recognition Techniques
• Derivatives
• Fourier Self-Deconvolution
• Curve Fitting
T
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)
T
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
T
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
T
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
T
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
T
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
T
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
T
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
T
DNA IR Marker BandsDNA IR Marker Bands
T
DNA Base IR
Differentiation of Cytidine (a,b) andmethyl derivativeswith FTIR
T
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
T
LIPIDS IR Marker BandsLIPIDS IR Marker Bands
T