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 Ca-D Proline
– 13C NMR of Proline
– Calculations of Proline Conformations
• Conclusion/Future Research
-C-D
Protein IR Spectrum
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
Ca-D bonds report on ψ/Φ dihedral changes
Deuterium labeling produces essentially no perturbation
Cα
D
• 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
Infrared (IR)
Raman
Pro/Con
Does 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/Pro
Spectral 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.
• 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 mm 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
-3
x 10
Absorbance
3
Solid:
2245 2202 2140 2060
At least 4 absorptions
Solution: 2240 2199 2135 2050
2
1
0
2250
2200
2150
2100
2050
2000
-1
Wavenumber (cm )
Similar to solid sampling, at least four absorptions related
to Pro-a-d1 were observed at approximately 2240, 2199,
2135, and 2050 cm-1
d
b
a
c
e
c
e
d
b
a
180
160
140
120
100
80
60
40
ppm
D
Hsyn
Cβ
Cα
• B3LYP/6-31G* calculations with
anharmonic corrections (Gaussian
03) were used to explore
frequency changes
• Protonated proline was used as a
model
C'
D-Cα-Cβ-Hsyn
Dihedral
D-Cα-C′-OH
• The D-Ca-Cb-Hsyn and D-Ca-C'-OH
Dihedral
dihedrals capture the ring flip and
CO2H rotation respectively
5.2 kcal/mol
5.4 kcal/mol
ring flip
2239 cm-1
2225 cm-1
CO2H
rotation
CO2H
rotation
ring flip
0.5 kcal/mol
0 kcal/mol
2246 cm-1
2244 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.
A combination band
at 2255 cm-1 was
predicted to be in
resonance with the
Ca-D stretch for all
four conformations.
CH2 / NH2 twisting
and D-Ca-N bend
CH2 / NH2 twisting
and H-O-C' bend
1035 cm-1
1220 cm-1
These calculations predict possible resonances associated with
CH2 / NH2 twisting and/or CH2 / NH2 twisting overtones.
-3
x 10
Absorbance
3
At least 4 absorptions
2
1
0
2250
2200
2150
2100
2050
2000
-1
Wavenumber (cm )
• 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.
D
O
D
O
D
O
-
D
-
O
+
NH
D
D
D
NH3
+
D
D
NH3
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).
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•
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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