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Exploring Protein Intramolecular Contact Rates and Protein Folding
Stewart N. Loh, Associate Professor of Biochemistry and Molecular Biology, SUNY Upstate Medical University.
Funding: NIH/NIGMS 5R01GM069755 (12/03-7/09).

One proposed model for protein folding is the hydrophobic collapse model, in which secondary structures are first formed before chains are drawn into their tertiary structure. Even in their denatured states, proteins appear to adopt residual secondary structures, and therefore it is important to study the rates at which the secondary structures contact each other, as this determines the limiting rate at which a protein can fold. In this project we use FCS to measure and characterize the fast and transient contacts made between different amino acids within the same protein that are crucial in guiding the protein to its folded conformation. We have measured the equilibrium dynamics of apomyoglobin (apoMb) labeled on the N-terminus with Alexa-488 over a range of pH from pH 2.6 where it is unfolded, to pH 6.3 where it is folded. We are particularly interested in data near pH 4.1, where a molten globule state is populated. Within our window of observation in the microsecond range, we were able to resolve three separate timescales on which the N-terminus makes transient contacts with specific amino acids elsewhere on the protein that quench its fluorescence (Chen et al 2007). The dynamics ranging from 3μs to 200μs reflect the conformational fluctuations that take place, even in the folded state of the protein.

The current collaboration has two major directions; the first being to sample a broader range of timescales which we will accomplish by slowing down the diffusion of the apoMb molecules so that they spend a longer time in the focal volume, increasing the range of observable dynamics into the millisecond domain. To accomplish this, we will trap individual apoMb molecules in lipid vesicles. The second direction involves the use of genetic mutations that will isolate specific amino acids responsible for the quenching.

References

Chen, H. E. Rhoades, J.S. Butler, S.N. Loh and W.W. Webb, 2007. “Dynamics of Equilibrium Folding Fluctuations of Apomyoglobin Measured by Fluorescence Correlation Spectroscopy,” PNAS 104(25), 10459-10464

 

 

 

 

 



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