The unfolding dynamics of hemoglobin (Hb) and human serum albumin (HSA) have been studied via Fluorescence Correlation Spectroscopy (FCS). This single-molecule technique may be used to elucidate protein unfolding pathways and, in particular, identify potential folding intermediates which are often hidden in ensemble measurements. The fluorescence fluctuations of Alexa Fluor 488-labeled Hb and HSA were observed under various denaturing conditions. The auto-correlation of these fluctuations provides information on the dynamics of protein diffusion and structural changes; thus fitting routines can be used to determine the diffusion time of the labeled proteins. The diffusion time may then be used to calculate the hydrodynamic radius, and thereby follow the protein expansion upon denaturant-induced unfolding. Therefore, FCS experiments allow for an understanding of the conformational dynamics of Hb and HSA, which can provide significant insight into the functional flexibility which is required of these proteins in the body.

Using fluorescence correlation spectroscopy to decipher the folding pathway of multi-domain proteins

243rd National Meeting (2012)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.