Bio-Applications of Vibrational Spectroscopy Biggest field – proteins and peptides a) Secondary structure Amide modes

C NO

H

C N

O

H

C N

O

H

I II III

~1650 ~1550 ~1300 IR – coupling changes with conform (typ. protein freq.) I II helix ~1650

+ 1550 must avoid H2O bend

sheet ~1630- 1530 often use D2O, but

coil ~1640-50 1520-60 lose amide II

Raman -see I, III – III has characteristic mix with CH

Depends on angle, characterize 2nd struct.

Raman inten-sity pattern changes - amide modes are coupled polymer tran-sitions. More side-chain modes

b) Active sites - structurally characterize, selective i) difference spectra – e.g. flash before / after - kinetic amides – COO

- / COOH – functional group

ii) Resonance Raman – intensify modes coupled to chromophore (e.g. heme)

Nucleic Acids – less use - helicity all about the same a) – monitor ribose conformation b) – single / duplex / triplex / quad – H-bond link bases

Sugars – little done, spectra broad, some branch appl. Lipids – monitor order – self assemble – polarization

Example is CH2 wag, but also stretch and scissor bend are characteristic Self assemble to lipid bilayer – membrane Polarization can tell orientation of lipid or protein in membrane

Amide A (N-H stretch) changes with H-bond, detect helix

Changes monitored, ProI has cis amide, ProII has trans Can also do thermodynamic studies of stability, helix-coil And get more detail, site-specific, by isotope labeling

13

C on amide C=O shift amide I frequency down ~40 cm-1

Dynamics, use stop-flow or T-jump for fast kinetics

-hairpin example T-jump

Isotopes give way to kinetic change by sitemechanism

Example Raman spectra of proteins (amide I not corrected for H2O interference) sharp bands often aromatic

Resonance Raman correlations (208 nm amide excitation)

DNA example IR spectra, RNA differ by base and ribose

Tautomerism identified by IR

Review: Discussed Diatomic Vibrations at length Polyatomics

a) expand V(q) = V(qe) + i

(V/qi)qi + j,i

(V/qiqj)qi qj

b) diagonalize V(q) V(Q) Qi =

j,i

cij qj linear combination x y z

on each atom; , … Normal coordinates

6 – Translations, rotations no potential E

eigen value “0” {diagonalize potential

(3N – 6) – vibrations internal nuclear motion examples: Triatomics

linear (1354) symmetric

OCO (2396) asymmetric

OCO )673( bend

OCO

bent

3825 3936 1654

O

H HH

O

H

O

H H

Biological uses key to characteristic frequency-intensity patterns, opens up many applications