Record Details

Studies of Protein Folding and Misfolding Using Fluorescence Correlation Spectroscopy (FCS) and other Biophysical Methods

EPrints@IICB

View Archive Info
 
 
Field Value
 
Title Studies of Protein Folding and Misfolding Using
Fluorescence Correlation Spectroscopy (FCS) and
other Biophysical Methods
 
Creator Basak, Sujit
Chattopadhyay, Krishnananda
 
Subject Structural Biology & Bioinformatics
 
Description Parkinson‟s disease (PD) is a multifactorial movement disorder (resting of tremor, rigidity,
Bradykinesia), in which both genetic as well as environmental factors play important roles.
Several evidences have been accumulated which implies that the aggregation of α-synuclein is a
critical factor in PD manifestation. Rare familial cases of PD have been associated with
mutations in α-synuclein even though the function of α-synuclein is still completely unknown.
The primary structure of purified α-synuclein, exists as a “natively unfolded” protein although a
recent report raises some controversies over the native structure of protein. A number of different
conformational states of α-synuclein including partially-folded (key intermediate in aggregation
and fibrillation), oligomeric species, as well as fibrillar and amorphous aggregates have been
observed. A number of factors that either accelerate or inhibit the rate of fibrillation have been
observed in vitro. Accelerators include environmental factors (certain pesticides and metals),
molecular crowding and various natural and synthetic charged polymers. α‑synuclein (α‑syn)
aggregation can take place either in the cytoplasm or when in association with the cellular
membrane. In the cytosol, unfolded monomers interact to form oligomers of varying
morphologies that eventually gets converted in to fibrils. The accumulation of these amyloid
fibrils leads to the formation of intracellular inclusions called Lewy bodies. Membrane bound
monomeric α‑syn adopts a predominantly α‑helical conformation and it undergoes a
conformational change via oligomerization to eventually form membrane bound β‑sheet‑rich
structures that self‑associate to form trans‑membrane amyloid pores and fibrils. During α‑syn
fibrillogenesis and aggregation, the intermediate species (oligomers and amyloid fibrils) are
highly toxic, affecting mitochondrial function, endoplasmic reticulum–Golgi trafficking, protein
degradation and/or synaptic transmission, all of which are thought to induce the process of
neurodegeneration.
We have aimed to establish the aggregation mechanism of alpha synuclein in membrane as well
as in cytosol. With the help of mainly single molecule fluorescence spectroscopy, a number of
heterogeneous intermediates are indentified in the presence of membrane or membrane mimics.
In this case, the secondary and tertiary structural changes, dynamic fluctuations (extended and
compact), and accumulation of aggregation prone species are observed in order to understand the
structural features and contribution of such conformations in the formation of amyloid
aggregates. Interestingly, the aggregation mechanism and the structural features of amyloid
fibrils in WT and familial mutants of alpha synuclein are different. This indicates that there is a
correlation between conformational changes in intermediates and the rate of aggregation.
But in the case of cytosol, the stability and aggregation mechanism of a protein cannot be
measured with the help of conventional methods as the viscosity and available space (i.e.;
excluded volume) severely alters and hence affects the single molecule detection. Here we have
developed a strategy that employs „two-diffusion fit‟ and „effective medium model‟ to evaluate
the conformation of proteins in the presence of viscous medium like polyacrylamide gel. With
the help of this model, we were able to measure the conformation of proteins with accuracy even
under viscous conditions. We could also explain the stability of protein as a function of excluded
volume effect. The structural deviation of a-syn from that of globular proteins have been shown
to deviate more under viscous conditions than under solution conditions, which could be
explained more clearly by using this method. The proper monitoring of heterogeneous
conformations that are generated during the aggregation process of alpha synuclein in membrane
and cytosol could be enabled which may pave way for newer therapeutics applications and may
help in revealing the mystery of protein stability in cellular environments.
 
Date 2015
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2327/1/THESIS__SUJIT_BASAK.pdf
Basak, Sujit and Chattopadhyay, Krishnananda (2015) Studies of Protein Folding and Misfolding Using Fluorescence Correlation Spectroscopy (FCS) and other Biophysical Methods. PhD thesis, University of Calcutta.
 
Relation http://www.eprints.iicb.res.in/2327/