| Synuclein Synuclein is a 140 residue natively unfolded protein implicated in Parkinson’s disease. Synuclein project involves following projects i) Copper binding to synuclein: identification of binding sites and their possible role in fibrillation. Involvement of heavy metals as causative agents for neurodegenerative disease has been reported in various recent articles. Various mechanisms, from oxidation of protein by the metal to the changes in the protein conformation, have been proposed to explain the effect of these metals. Synuclein (syn) family (a, b and g) - causing Parkinson’s disease (PD) upon fibrillation, has been found to be very sensitive to metals. In this study, we have systematically tried to recognize the a-syn-Cu2+ and b-syn-Cu2+ binding sites first and then look into the possible role of metal binding towards fibrillation using electron spin resonance (ESR), electron spin-echo envelope modulation (ESEEM), circular dichroism (CD) and peptide models. Our results suggest that there are at least two different Cu2+ binding sites in both a- and b-syn’s each having a different affinity for Cu2+ with not a very significant difference in overall equivalence ratio of Cu2+-syn binding for both systems. ESR of two peptide fragments (N-terminal and His-50 containing peptides) containing suspected binding sites show similar signature as in intact proteins but ESEEM studies favor predominance of N-terminal binding over the His binding except for His-50 containing peptide. Near UV CD studies show a concurrent change in tertiary structure upon copper binding to both the syn’s. It is more likely, therefore, that copper binding causes structural changes in syn’s making them more susceptible to fibrillation leading to PD. Ahmad, A: Burns ,CS: and Fink AL. Binding of Copper and its effect on the fibrillation of alpha synuclein. (Manuscript under preperation) ii) Interaction of synuclein with heat shock proteins: Heat shock proteins Dna K, Dna J and GrpE have been reported to help proper folding and/or degradation of misfolded proteins. In combination with the ATP, 5mM MgCl2 and 200mM KCl The first step has been found to be the binding of the nascent chain to Dna K e.g. RCMLA (reduced carboxymethylated lactalbumin). The hypothesis is that every protein has a Dna K binding site after every 36th amino acid. We found that synuclein does not bind to the Dna K at least in the way and extent of RCMLA. Instead synuclein showed a aggregation peak in HPLC profile that unlike the RCMLA adduct peak did not dissociate with ATP. The fibrillation showed faster rates in presence of Dna J alone whereas the combination provided stability against fibrillation. The studies were repeated in presence of 20% glycerol to mimic the crowded conditions of the cell environment. Ahmad, A: Sargenti, C: and Fink, AL. (2003) Interaction of synuclein with heat shock proteins (2003) (Manuscript prepared). iii) Mutants: I have made the mutant A140C of synuclein for SH labeling by various probes and other mutants where Cysteine will be introduced at various places is going on. The goal of this project is to locate the regions of the synuclein that come into contact first and follow the process of stacking of the synuclein molecules into fibrils. |