Single molecule fluorescence correlation spectroscopy (FCS) can be used to determine the specific folding pathway of a fluorescently labeled protein. Here, Alexa-Fluor 488 labeled human serum albumin (HSA) was characterized using FCS. Human serum albumin is a protein with three domains that is abundant in human blood plasma and has a multitude of functions such as transporting hormones and fatty acids. This model protein was studied via FCS in a range of denaturant concentrations to identify occupied intermediate states along its folding pathway. A truncated HSA containing only domain 1 was also studied. The FCS data was analyzed to extract the diffusion time associated with the protein molecules. The diffusion time can be used to calculate the radius of hydration of the labeled protein which is related to the protein conformation. Identifying the conformations of both the full-length HSA and of only domain 1 in different denaturant concentrations allows us to gain a better understanding of the specific folding pathway undergone by HSA during denaturation.

Studying the folding pathway of truncated and full-length human serum albumin via FCS

248th National Meeting (2014)

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