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Studies of Protein Conformation and Dynamics Using Fluorescence Correlation Spectroscopy (FCS) and Other Biophysical Methods

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Title Studies of Protein Conformation and Dynamics Using Fluorescence Correlation Spectroscopy (FCS) and Other Biophysical Methods
 
Creator Halder, Subhasis
 
Subject Structural Biology & Bioinformatics
 
Description The mechanism by which the unfolded chain of a protein folds into its functional three
dimensional structure has been a matter of intense debate. Some of the major challenges in
protein folding are as follow: a) how do the initial contacts in the unfolded state modulate the
late stage of protein folding? b) How to design experimentation to study the inherent
heterogeneity of the protein folding pathway? This is important because the conventional bulk
spectroscopic measurements have been found inadequate to monitor the intricacies of protein
folding pathways. c) How to study the mechanism of aggregation and design inhibitor molecules
to block protein aggregation? This is because protein aggregation has been shown to have direct
relevance in a number of neurodegenerative diseases.
In this thesis, we have used fluorescence correlation spectroscopy (FCS) to address some of the
above questions. FCS measures fluorescence fluctuations in a small observation volume while
the system is kept under thermodynamic equilibrium. These fluctuations can originate either
from the molecular diffusion inside the observation volume or through any chemical kinetics or
conformational events. FCS is a unique experimental technique which can measure both, the
conformation of the protein molecule and its fluctuation dynamics in the μsec time scale at single
molecule resolution.
We have shown that FCS can be used to study the effect of a number of protein stabilizers on the
conformation and μsec dynamics of fluorescently labelled cytochrome c. Measuring ureainduced
unfolding of cytochrome c in the absence and presence of arginine, we show that
arginine inhibits the formation of a partially folded aggregation prone intermediate. Other
stabilizers, e.g. sucrose, NaCl, and proline, do not show this behaviour. Our result implicate that
the effect of arginine on protein self-association may have come from its interaction with side
chains, while a traditional osmolyte like sucrose influences only the protein backbone. To
validate this hypothesis, we have used surfactant self association as a “super simplified” model
of protein aggregation. Using two common surfactants, namely SDS and DTAB, we have
established the dual nature of arginine. In a separate study, we have shown that initial
hydrophobic collapse and the formation of secondary structure of a protein take place differently
depending on the solution conditions.
 
Date 2012
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1694/1/Final_thesis_Shubhasis_haldar.pdf
Halder, Subhasis (2012) Studies of Protein Conformation and Dynamics Using Fluorescence Correlation Spectroscopy (FCS) and Other Biophysical Methods. PhD thesis, Calcutta University.
 
Relation http://www.eprints.iicb.res.in/1694/