Studies Of Cytochrome c Folding And Conformational Dynamics Using Experimental And Computational Methods
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Studies Of Cytochrome c Folding And Conformational Dynamics Using Experimental And Computational Methods
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| Creator |
Paul, Simanta Sarani
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| Subject |
Structural Biology & Bioinformatics
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| Description |
Protein folds from an unstructured/unfolded state to a more organized compact structure in order to attain a functional form. The conformational search during the folding process is difficult to comprehend as the time taken by a polypeptide chain, to search the infinite number of possible conformations, is surprisingly fast. It has been found that, this complex search algorithm is governed by multiple parameters, which include; amino acid sequence, structural architecture, solvent conditions, interactions with other macromolecules like proteins or protein complexes and the presence of chaperones etc. It has been found that the folding energy landscape of a protein is like a funnel having rough surface, containing various kinetic traps. Depending upon the above mentioned parameters, the protein chain samples various minima in the folding energy landscape, while rolling down the funnel surface. The conformations at each minimum have varying degree of stability and in general, the functional form of a protein is relatively more stable as compared to others states, like intermediate states. It is to be noted that a single protein can attain various functional forms and these functional forms are not necessarily the most stable state of the protein. The most stable state of a protein is the aggregated state, and this end state can be achieved by the protein, in various routes. The normal or favored route is highly populated by functional forms and on-pathway intermediates help the folding flux towards the functional form. The structural degradation over time of the functional form, in-vitro, leads to the aggregation while in-vivo the degraded and nonfunctional forms can be taken care-off by protease actions. Nevertheless, the folding process is not error free; and peptide chain folding can be derailed from the normal route to form miss-folded species / off-pathway intermediates and ultimately leading to aggregation.
The overall folding process can be complex with respect to both execution mechanism and outcome possibilities. It has been found that the folding energy landscape can be complemented by binding energy landscape to generate stable states, which may have functional as well as non-functional aspects. The conformational heterogeneity and the associated dynamic variability of a protein contribute significantly to define its folding, ligand binding and aggregation landscape.
In this thesis, we have investigated the conformation and dynamic heterogeneity of a protein using cytochrome c as a model. To do this, we have studied the binding of cytochrome c, from higher eukaryote (horse) and lower eukaryote (yeast), with negatively charged phospholipid, cardiolipin. This investigation may have important implications in the context of the understanding of the secondary functions of cytochrome c, which become important during apoptosis. We have found that the secondary function of cytochrome c, specially the pro-apoptotic activity, varies among the source of cytochrome c. We have used fluorescence correlation spectroscopy to find out the presence of three separate conformers, having varying degree of brightness (a dark compact conformer, a bright expanded conformer and oligomers), which are in equilibrium with each other in the presence of cardiolipin. An understanding of the complex interplay within these three populations is crucial for the pro-apoptotic functions of these proteins. We found that cytochrome c from yeast fluctuates more compared to the cytochrome c from horse. As a result of this fluctuation, the yeast protein is peroxidase active in the native state, but horse cytochrome c requires the help of cardiolipin to become peroxidase active. In the presence of cardiolipin, cytochrome c from yeast becomes aggregation prone and its peroxidase activity decreases. We have concluded that, in the case of yeast cytochrome c the formation of non-functional aggregates could be responsible for its lack of pro-apoptotic activity. We have also identified the key residues that may be responsible for higher degree of fluctuations in yeast cytochrome c. Subsequently, we have explored the folding as well as cardiolipin binding landscape of yeast cytochrome c in synthetic crowded media. We have found that the presence of crowded media disfavors the expanded state populations of yeast cytochrome c. This behavior helps the protein to control the unwanted peroxidase activity inside a healthy yeast cell. Our study also shows that the favored compactness in yeast cytochrome c population destabilizes the protein. We have additionally reported that although the crowded media favors the compact conformer due to exclusion volume effect, the sub-folding rate between expanded state and compact state is affected by the micro-viscosity of the crowded media. In a separate work we have established that fluorescence correlation spectroscopy and molecular dynamic simulation can be used as a complementary technique to explore the unfolding of a truncated cytochrome c from bacteria (Pseudomonas aeruginosa) (cytochrome VIII
c551). By using these techniques, we have characterized the intermediate state of cytochrome c551 demonstrating the shape change of this protein, which occurs as a result of unfolding.
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| Date |
2016
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| Type |
Thesis
NonPeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/2776/1/Ph.D._thesis_SIMANTA.pdf
Paul, Simanta Sarani (2016) Studies Of Cytochrome c Folding And Conformational Dynamics Using Experimental And Computational Methods. PhD thesis, University of Calcutta. |
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| Relation |
http://www.eprints.iicb.res.in/2776/
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