Transcript
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
top related