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DEVELOPMENT OF RAMAN INSTRUMENTATION AND METHODOLOGIES FORPEPTIDE AND PROTEIN INVESTIGATIONS

Bykov, Sergei V. (2010) DEVELOPMENT OF RAMAN INSTRUMENTATION AND METHODOLOGIES FORPEPTIDE AND PROTEIN INVESTIGATIONS. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Raman spectroscopy is a tool which offers numerous advantages for investigating biological macromolecules and complex systems. The advantages include but are not limited to: ultrahigh sensitivity to changes in molecular bond lengths (better then pm resolution); time-resolved measurements down to picoseconds time intervals; high selectivity offered by resonance enhancement; high adaptivity for investigation of objects of various sizes in different physical state.This thesis is focused on the development of UV resonance Raman spectroscopy as a sensitive and incisive technique for polypeptide secondary structure determinations. We built a state-of-the-art tunable UV Raman spectrometer for the 193 - 270 nm spectral region. This instrument allows for steady state and transient UV Raman measurements of the conformational transitions of polypeptides and other macromolecules. We also continued our search for new polypeptide backbone conformational markers. For the first time we investigated the dependence of the CαH2 stretching vibrations frequencies on Ramachandran φ and ψ angles in glycine-based peptides and showed their potential for polypeptide conformational analysis.We investigated the conformational preferences of polyglycine, poly-L-lysine and poly-L-glutamic acid in aqueous solutions in their unfolded states. Our studies indicate that polyglycine in solution assumes a broad ensemble of conformations centered around the Ramachandran angles of the 3-1-helix. We explained this polyglycine conformational preference by favorable electrostatic interactions between adjacent peptide bond carbonyl dipoles. Poly-L-lysine and poly-L-glutamic acid in non-α-helical states in solution show a preference for a left-handed 3-1-helix conformation with some contribution from a left-handed 2.5-1-helical conformation which is likely stabilized due to electrostatic repulsion between charged side chains. In addition, we find that a poly-L-lysine and poly-L-glutamic acid mixture at neutral pH is ~60% β-sheet.We used time resolved UV resonance Raman spectroscopy to characterize the spatially resolved (termini vs. center) kinetics of thermal unfolding of a 21 amino acid isotopically labeled, mainly alanine, peptide. We found that the relaxation rates are significantly different for the middle and terminal peptide bonds and strongly depend on T-jump temperatures. We explain the observed kinetics in terms of different relative contributions of different α-helix-like motifs such as pure α-helices, π-bulges and 3-10 helices to the observed melting kinetics.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Bykov, Sergei V.bykov@pitt.eduBYKOV
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairAsher, Sanfordasher@pitt.eduASHER
Committee MemberChong, Lilian
Committee MemberWetzel, Ronald
Committee MemberSaxena, Sunil
Date: 16 June 2010
Date Type: Completion
Defense Date: 14 December 2009
Approval Date: 16 June 2010
Submission Date: 7 December 2009
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Spectroscopy; polypeptide structure determination; UV resonance Raman
Other ID: http://etd.library.pitt.edu/ETD/available/etd-12072009-203151/, etd-12072009-203151
Date Deposited: 10 Nov 2011 20:09
Last Modified: 15 Nov 2016 13:53
URI: http://d-scholarship.pitt.edu/id/eprint/10163

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