Continued from part a Characteristic Amide Vibrations A often - PowerPoint PPT Presentation

Continued from part a

Characteristic Amide Vibrations A – often obscured ~3300 cm -1 by solvent I - Most useful ; ~1650 cm -1 IR intense, less interference (by solvent, other modes,etc) Less mix ( with other modes) Also Raman 1500-50 cm -1 II - IR intense mix Not Raman, unless RR III - Raman Intense 1300-1250 cm -1 Weak IR Multiple bands IV – VII – difficult 700 cm -1 to detect, discriminate

Peptide conformation depends on f , y angles If ( f,y) repeat, they determine secondary structure Chromophores – amides are locally achiral CD has little signal without coupling, ideal for detection -- IR, Raman resolve shift Detection requires method sensitive to amide coupling Far UV absorbance broad, little fluorescence — coupling impact small

Model polypeptide IR absorbance spectra - Amide I and II I helix 3 II Absorbance 2  -structure (Not in Raman) 1 random coil 0 (weak IR but 1750 1700 1650 1600 1550 1500 1450 strong in Raman) Wavenumbers (cm -1 )

Combining Techniques: Vibrational CD “CD” in the infrared region Probe chirality of vibrations  goal stereochemistry Many transitions / Spectrally resolved / Local probes Technology in place -- separate talk Weak phenomenon - limits S/N / Difficult < 700 cm -1 Same transitions as IR same frequencies, same resolution Band Shape from spatial relationships neighboring amides in peptides/proteins Relatively short length dependence AA n oligomers VCD have D A/A ~ const with n vibrational (Force Field) coupling plus dipole coupling Development -- structure-spectra relationships Small molecules – theory / Biomolecules -- empirical, Recent — peptide VCD can be simulated theoretically

VIBRATIONAL OPTICAL ACTIVITY Differential Interaction of a Chiral Molecule with Left and Right Circularly Polarized Radiation During Vibrational Excitation VIBRATIONAL CIRCULAR DICHROISM RAMAN OPTICAL ACTIVITY Differential Absorption of Left and Right Differential Raman Scattering of Left Circularly Polarized Infrared Radiation and Right Incident and/or Scattered Radiation

UIC Dispersive VCD Schematic Yes it still exists and measures VCD! Electronics Pre- Dynamic Tuned    C D Amp Normalization Filter Lock-in Lock-in Transmission PEM ref.  M Feedback Lock-in Chopper ref.  C A/D G Monochromator Interface Computer Interface Optics and Sampling C F S M2 M1 L D SC P PEM

Separate VCD Bench Optics UIC FTIR FT-VCD Schematic (designed for magnetic VCD commercial ones simpler ) Electronics Polarizer PEM (ZnSe) detector Sample FT-computer Optional magnet filter Detector (MCT) lock-in amp PEM ref

Large electric dipole transitions can couple over longer ranges to sense extended conformation Simplest representation is coupled oscillator     π    ) m a    m  m T ab R    T ab a b  2  m b c De  Dipole coupling results in a e L -e R l derivative shaped circular dichroism Real systems - more complex interactions - but pattern is often consistent

Selected model Peptide VCD, aqueous solution Amide I Amide II 30 helix D A a 20 VCD (A. U.)  10  -structure 0 random coil coil -10 1750 1700 1650 1600 1550 1500 1450 Wavenumbers (cm-1)

Nature of the peptide random coil form 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 Dukor and Keiderling 1991 with ECD, VCD, and IR showed Pro n oligomers 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

ECD of Pro n oligomers Reference: Poly(Lys) – “coil”, pH 7 Single amide Builds up to sheet Poly-Pro II ‘coil’ frequency --> helix tertiary amide Dukor, Keiderling - Biopoly 1991 Greenfield & Fasman 1969

Relationship to “random coil” - compare Pro n and Glu n IR ~ same, VCD - same shape, half size -- partially ordered Dukor, Keiderling - Biopoly 1991

Thermally unfolding “random coil” poly -L-Glu -IR, VCD T = 5 o C ( ___ ) VCD loses 25 o C (- - -) magnitude 75 o C (-.-.-) IR shifts “random coil” frequency must have local order Keiderling. . . Dukor, Bioorg-MedChem 1999

Comparison of Protein VCD and IR FTIR in H 2 O VCD in H 2 O 2 a HEM HEM CON  D A CON A LYS a/ LYS 1700 1650 1600 1550 1500 1700 1650 1600 1550 1500 Wavenumbers (cm -1 ) Wavenumbers (cm -1 )

VCD Example: a - vs. the 3 10 -Helix a -Helix 3 10 -Helix i, i+4  H-bonding  i, i+3 3.6  Res./Turn  3.0 2.00  Trans./Res (Å)  1.50

The VCD success example: 3 10 -helix vs. a -helix i -> i+3 4 310-helical 500 Aib 2 LeuAib 5 400 3 10 3 A 300 Ala(AibAla)3 Absorbance D A (A.U.) 200 2 (Aib-Ala)6 100 mixed 1 a 0 a (Met 2 Leu) 6 a -helical i -> i+4 -100 0 1800 1600 1400 1800 1600 1400 Wavenumbers (cm-1) Wavenumbers (cm -1 ) Relative shapes of multiple bands distinguish these similar helices Silva et al. Biopolymers 2002

Simulated IR and VCD spectra The best practical computations for the largest possible molecules 1. Ab Initio (DFT) quantum mechanical calculations can give necessary data for small molecules Frequencies from force field -diagonalize second derivatives of the energy Intensities from change in dipole moment with motion Express all as atomic properties 2. Large bio-macromolecules --need a trick (Bour et al. JCompChem 1997) Transfer atomic properties from “small” model In our case these “small” calculations are some of the largest peptides ever done ab initio

Transfer of FF, APT and AAT (e.g. Ala 7 to Ala 20 ) Method from Bour et al. J. Comp Chem. 1997 Main chain residues 20-mer Middle C-terminus N-terminus residue 7-mer: FF, APT, AAT calculated at BPW91/6-31G* level Kubelka, Bour, et al., ACS Symp. Ser .810, 2002

Uniform long helices  characteristic, narrow bands 2 d a Simulations 0 -2 7-amide disperse -4 2 amide I, II bands a d vacuum 3 D 2 O 2 De 1 21-amide: narrow 3 IR band by change 2 a d 0 intensity distribution, e -2 preserve mode -4 De dispersion and VCD 3 a d shape, solvent -- 2 De 1 close amide I-II gap 1800 1700 1600 1500 1400 1300 1200 1800 1700 1600 1500 1400 1300 1200 Kubelka & Keiderling, Wavenumber (cm -1 ) Frequency error mostly solvent origin J.Phys.Chem.B 2005 e

Simulation of Helix IR and VCD Really Works! Experiment: Simulation: 3 10 -helix 3 10 -helix vs. a -helix: comparison of Aib n , Aib Ac-(Aib) 8 -NH 2 -Leu-Aib 2 5 Ala n and (Aib-Ala) n sequences . Simulation: a -helix Ac-(Aib-Ala) 4 -NH 2 (Aib-Ala) in TFE Ac-(Aib-Ala) 3 -NH 2 4 in CDCl De /amide De /amide Ac-(Ala) 8 -NH 2 (Met -Leu) Ac-(Ala) 6 -NH 2 2 8 1700 1600 1500 1700 1600 1500 1700 1600 1500 -1 ] -1 ] -1 ] Wavenumber [cm Wavenumber [cm Wavenumber [cm (Kubelka,Silva, Keiderling JACS 2002)

Isotopic Labeling – old technique - new twist Shift frequency by  ~ (k/ m ) 1/2 Tends to decouple from other modes, and can disrupt their exciton coupling Not intense, compare to polymer repeat Isolated oscillator (transition) in other modes Requirement: High S/N, good baseline focus on one band  dispersive VCD?

a -helix model: Alanine 20-mer 13 C labeling scheme Notation Label position Peptide sequence unlabeled none Ac-AAAAKAAAAKAAAAKAAAAY-NH 2 Ac- AAAA KAAAAKAAAAKAAAAY-NH 2 L1 N-terminus Ac-AAAAK AAAA KAAAAKAAAAY-NH 2 L2 Middle (closer to N-terminus) L3 Middle (closer to C-terminus) Ac-AAAAKAAAAK AAAA KAAAAY-NH 2 L4 C-terminus Ac-AAAAKAAAAKAAAAK AAAA Y-NH 2 Silva, Kubleka, et al. PNAS 2000

a -helix ProII-like Unlabeled Unlabeled N-terminus N-terminus Simul. C-terminus C-terminus 4 Middle (N) Middle (N) Middle (C) Middle (C) e  x 10 -3 ) 2 1750 1700 1650 1750 1700 1650 High T Unalbeled Unlabeled Low T N-terminus 12 N-terminus C-terminus C-terminus Middle (N) Middle (N) Middle (C) Middle (C) Exper. A norm ( x 10) 8 4 0 1700 1650 1600 1550 1700 1650 1600 1550 Wavenumber [cm -1 ] Wavenumber [cm -1 ] Simulated and experimental IR absorption for Ala 20 with 13 C labels C-term is different, do not know structure from IR Silva, Kubleka, et al. PNAS 2000

2 a -helix ProII-like 0 De  x 10) -2 -4 Unlabeled Unlabeled N-terminus N-terminus C-terminus -6 C-terminus Middle (N) Middle (N) Middle (C) Middle (C) -8 1750 1700 1650 1750 1700 1650 High T 4 Low T 5 ) 0 D A norm ( x 10 -4 Unlabeled Unlabeled N-terminus N-terminus -8 C-terminus C-terminus Middle (N) Middle (N) Middle (C) Middle (C) 1700 1650 1600 1550 1700 1650 1600 1550 Wavenumber [cm -1 ] Wavenumber [cm -1 ] Simulated and experimental VCD for Ala 20 with 13 C labels VCD shows helical at all but C- terminal, where it is “coil” Silva, Kubleka, et al. PNAS 2000

4 a b 0 D A (x10 5 ) 5 deg 10 deg -4 15 deg 20 deg 25 deg 30 deg 35 deg -8 40 deg 45 deg 50 deg 4 55 deg 60 deg c d 0 D A (x10 5 ) -4 -8 1660 1620 1580 1660 1620 1580 Wavenumber [cm -1 ] Temperature dependent Ala 20 VCD: a) unlabeled b) C-terminus c) N-terminus d) Middle(N) labeled