Download - Andrew Durgan Department of Chemistry & Biochemistry Gonzaga University September 24, 2009
• Introduction– Carbon-Deuterium Labeled Amino Acids– Methods: Vibrational Techniques– Infrared Spectroscopy (IR)
• IR versus Raman Spectroscopy• IR Sampling
• Results– IR of C-D Proline
– 13C NMR of Proline– Calculations of Proline Conformations
• Conclusion/Future Research
-C-D
O-H, C-H, N-H Amide bands & C=C, C=O, C=N
• C-D frequency shifted away from unlabeled vibrations• Site-specific isotopic labeling improves structural resolution• C-D bonds report on their local environment• C-D bonds report on ψ/Φ dihedral changes• Deuterium labeling produces essentially no perturbation
Protein IR Spectrum
• Unique ring structure imposes backbone conformational constraints
• Proline appears in many neuropeptides• cis-trans Amide isomerization is hypothesized to play a role in
neuropeptide selectivity
D
Cα
Raman
Pro/ConDoes not cause local heating or photodecomposition of the sample
Can cause local heating or photodecomposition of the sample
More accessible instrumentation Less accessible instrumentation due to laser requirement
Con/ProSpectral observations can be obscured by H2O (g) and CO2 (g) absorptions
Spectral observations are not obscured by H2O (g) and CO2 (g) absorptions.
Conclusion: Both spectroscopic techniques can be useful to C-D labeled amino acid investigations.
Infrared (IR)
•Potassium Bromide Pellets
2 mg of sample were homogenized with 80 mg of KBr
•Transmission Spectra
20 µL of aqueous samples (0.050 – 1.0 M) were dispensed between CaF2 plates with a 75 m
spacer.
•Scan Parameters
The sample chamber was purged with N2 and 8000
scans at 4 cm-1 resolution were collected using an MCT detector.
At least four absorptions related to Pro-a-d1 were
readily observed at approximately 2245, 2202,
2140, and 2060 cm-1
2000205021002150220022500
1
2
3
x 10-3
Wavenumber (cm-1)
Absorb
ance
At least 4 absorptions
2000205021002150220022500
1
2
3
x 10-3
Wavenumber (cm-1)
Absorb
ance
At least 4 absorptions
Similar to solid sampling, at least four absorptions related to Pro--d1 were observed at approximately 2240, 2199,
2135, and 2050 cm-1
Solid: 2245 2202 2140 2060
Solution: 2240 2199 2135 2050
D-Cα-C′-OHDihedral
D-Cα-Cβ-Hsyn
Dihedral
Cβ Cα
Hsyn
D
C'
• B3LYP/6-31G* calculations with anharmonic corrections (Gaussian 03) were used to explore frequency changes
• Protonated proline was used as a model
• The D-C-C-Hsyn and D-C-C'-OH dihedrals capture the ring flip and CO2H rotation respectively
CO2H rotation
CO2H rotation
ring flip
ring flip
5.2 kcal/mol
2225 cm-1
0 kcal/mol
2244 cm-1
0.5 kcal/mol
2246 cm-1
5.4 kcal/mol
2239 cm-1
Relative electronic energies and C-D stretching frequencies are presented. For zwitterionic Pro, only two conformations might be expected due to
symmetry of the carboxylate group.
CH2 / NH2 twisting and H-O-C' bend
1220 cm-1
CH2 / NH2 twisting and D-C-N bend
1035 cm-1
A combination band at 2255 cm-1 was predicted to be in
resonance with the C-D stretch for all
four conformations.
These calculations predict possible resonances associated with CH2 / NH2 twisting and/or CH2 / NH2 twisting overtones.
• Pro ring flipping is faster than the NMR timescale but not the IR timescale, which is consistent with the observation of multiple IR absorptions and only one set of NMR signals.
• Calculations predict that the observed absorption bands arise from Pro ring flipping, resonances associated with CH2 / NH2 twisting, and possible CH2 / NH2 twisting overtones.
• Specific assignments are in progress.
2000205021002150220022500
1
2
3
x 10-3
Wavenumber (cm-1)
Absorb
ance
At least 4 absorptions
2000205021002150220022500
1
2
3
x 10-3
Wavenumber (cm-1)
Absorb
ance
At least 4 absorptions
O
NH3+
O-
D
D
D
D
D
O
NH3+
NH
O-
D
D
D
D
D
• Trp & Phe show absorptions at 2310 / 2280 & 2275 cm-1
• Future work includes incorporating C-D labeled amino acids (Pro, Phe, & Trp) into neuropeptides (e.g. Try-Pro-Trp-Phe).
• Dr. Matthew Cremeens, Gonzaga University• Christy Watson, Gonzaga University• Matt Deslman, Gonzaga University• Dr. Steven Corcelli, Notre Dame• Gonzaga Science Research Program• Howard Hughes Medical Institute