Dahlburg, Elizabeth M.
(2016)
Primary Amide Raman Vibrations as Environmental and Structural Markers of Glutamine and Asparagine Protein Side Chains.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
UV resonance Raman (UVRR) is a powerful spectroscopic technique for the study of protein conformation and dynamics. Excitation at ~200 nm selectively enhances secondary amide vibrations, which are sensitive to the peptide backbone secondary structure and local environment. Primary amide bands are also resonance enhanced in UVRR spectra and could be used to report on glutamine and asparagine side chains in biophysical studies of proteins and peptides.
IR absorption, visible Raman, and UVRR were used to investigate the small primary amide molecule propanamide. Dramatic spectral changes in the primary amide vibrations were observed upon aqueous solvation. Aqueous solvation impacts the dielectric and hydrogen bonding environment of the primary amide group resonance structures. This leads to a decrease in C--O and increase in C--N bond order of the primary amide group and therefore alters the resonance enhancement and vibrational frequencies of the primary amide vibrations substantially. Due to this significant response, several primary amide bands can be used as sensitive environmental markers for the glutamine and asparagine side chains.
Visible Raman and UVRR spectra of L-glutamine and five derivative molecules, D-glutamine, N-Acetyl-L-glutamine, L-glutamine t-butyl ester, Glycyl-L-glutamine, and L-seryl-L-asparagine, were collected and assigned in the 950-1200 cm-1 region. The OCCC dihedral angle of each was determined from X-ray crystal structures. An empirical relationship between the AmIIIP vibrational frequency and the OCCC dihedral angle was observed. This dependence can be explained by hyperconjugation that occurs between the Cbeta--Cgamma sigma orbital and the C=O pi* orbital of the primary amide group. This interaction induces an increase in the Cbeta--Cgamma bond length. As the Cbeta--Cgamma bond length increases, the stretching force constant decreases, downshifting the AmIIIP band. Due to this sensitivity, the AmIIIP can be used as a structural marker diagnostic of the OCCC dihedral angle, such as in the side chains glutamine and asparagine in peptide and protein conformational studies.
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| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
20 June 2016 |
| Date Type: |
Publication |
| Defense Date: |
3 March 2016 |
| Approval Date: |
20 June 2016 |
| Submission Date: |
1 April 2016 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
62 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Molecular Biophysics and Structural Biology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Raman, UV resonance Raman, glutamine, protein structure, side chain |
| Date Deposited: |
20 Jun 2016 20:27 |
| Last Modified: |
15 Nov 2016 14:32 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/27554 |
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