Dynamic Aspects of Protein Structure - a Fluorescence Spectroscopic Approach
Marriott, Gerard Joseph
This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.
Permalink
https://hdl.handle.net/2142/70562
Description
Title
Dynamic Aspects of Protein Structure - a Fluorescence Spectroscopic Approach
Author(s)
Marriott, Gerard Joseph
Issue Date
1987
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Structural fluctuations of proteins were investigated using steady state and time-resolved fluorescence techniques. High resolution anisotropy decay measurements were used to test the findings of molecular dynamics calculations on tryptophan and tyrosine residues in lysozyme and bovine pancreatic trypsin inhibitor (BPTI) respectively. Anisotropy decays measured over a large viscosity range failed to observe the very fast rotational modes of intrinsic fluorophores calculated in the molecular dynamics approach. The experimental rotational correlation times for the tryptophan residues in lysozyme were approximately 1 nanosecond at room temperature in aqueous solution and approximately 0.6 nanosecond for the tyrosine residues in BPTI.
The range of rates and amplitudes of protein structural fluctuations were used as a yardstick for those motions observed using indirect fluorescence methods of protein dynamics. Using this technique and considering a number of solvent properties the rates of dipolar relaxation in toluene, methanol and propylene glycol were determined. Dynamic relaxation processes were seen in propylene glycol at viscosities in excess of 10('4) poise indicating that gross rearrangement of the solvent dipole is not required. The involvement of ground state effects such as hydrogen bonding are discussed in relation to this finding.
With a suitable meter to observe dynamic solvent relaxation the same type of events were studied in proteins specifically labelled with PRODAN. Fluorescent conjugates of PRODAN in human serum albumin, bromelain and papain failed to demonstrate dynamic dipolar relaxation on the nanosecond timescale over a viscosity range of 1-10('4) poise. The discrepancy between these two results lies in the nature of the absorption band which is quite complex and is typically non-Gaussian in energy. The precision in the determination of the ground state energy is therefore at or beyond the relaxation energies determined from steady state approaches. Time-resolved measurements therefore offer an unambiguous method of describing the presence and rates of dipolar relaxation. The absence of dipolar relaxation events in the proteins is discussed.
Finally the electrostatic interaction energy between PRODAN and papain was calculated using simple electrostatic considerations. A computer synthesized PRODAN-papain conjugate was analyzed and electrostatic contributions from peptide dipoles, surface water and changes determined. (Abstract shortened with permission of author.)
Use this login method if you
don't
have an
@illinois.edu
email address.
(Oops, I do have one)
IDEALS migrated to a new platform on June 23, 2022. If you created
your account prior to this date, you will have to reset your password
using the forgot-password link below.
