Thermal and denaturation studies of the time-resolved fluorescence decay of superoxide dismutase
Silva, Norberto De Jesus
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/2142/28690
Description
Title
Thermal and denaturation studies of the time-resolved fluorescence decay of superoxide dismutase
Author(s)
Silva, Norberto De Jesus
Issue Date
1993
Doctoral Committee Chair(s)
Gratton, E.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
human superoxide dismutase
fluorescence decay
Language
en
Abstract
Time-resolved fluorescence of single tryptophan proteins have demonstrated the complexity of protein dynamic and structure. In particular, for some single tryptophan proteins, their fluorescence decay is best described by a distribution of fluorescence lifetimes rather than one or two lifetimes. Furthermore, the behavior of the lifetime distributions with environmental changes is consistent with the hypothesis that proteins fluctuate between a hierarchy of many conformational substates. With this scenario as a theoretical framework, the correlations
between protein dynamic and structure are investigated by studying the time-resolved fluorescence and anisotropy decay of the single tryptophan (Trp) residue of human superoxide dismutase (HSOD) over a wide range of temperatures and at different denaturant concentrations. First, it is demonstrated that the center of the lifetime distribution can characterize the average deactivation environment of the excited Trp-protein system. A qualitative model is introduced to explain the time-resolved fluorescence decay of HSOD in 80% glycerol over a wide range of temperatures. The dynamical model features isoenergetic conformational substates separated by a hierarchy of energy barriers. The HSOD system is also investigated as a function of denaturant concentration in aqueous solution. As a function of guanidine
hydrochloride (GdHCl), the width of the fluorescence lifetime distribution of HSOD displays a maximum which is not coincident with the fully denatured form of HSOD at 6.5M GdHCl. Furthermore, the width for the fully denatured form of HSOD is greater than that of the native form. This is consistent with the scenario that more conformational substates are being created upon denaturation of HSOD. HSOD is a dimeric protein and it was observed that the width of the lifetime distribution of HSOD at intermediate GdHCl concentrations increased with decreasing protein concentration. In addition, the secondary structure of HSOD at intermediate GdHCl concentration does not change with protein concentration. These results suggest that HSOD display structural microheterogeneity which is consistent with the hypothesis of conformational substates. Further analysis show that, during denaturation, the monomeric form of HSOD is an intermediate which displays native-like secondary structure and :fluctuating tertiary structure; i.e., the monomeric form of HSOD is a molten globule.
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.
