The conformational dynamics of human serum albumin (HSA) were investigated via fluorescence correlation spectroscopy (FCS) to gain insight into the protein folding mechanism.  Alexa Fluor 488 maleimide, was used to label Cys-34 in domain I for detection by the FCS setup. From analysis of the autocorrelation of the fluorescent bursts caused by diffusion of the labeled protein in and out of the confocal volume, information regarding the diffusional properties of the protein could be determined.  Specifically, the hydrodynamic radius of HSA as a function of guanidine hydrochloride concentration was calculated using the Stokes-Einstein equation. As expected, the denaturation of HSA by the guanidine hydrochloride yielded an increase in labeled HSA diffusion time. As FCS is capable of identifying transient intermediates which are concealed by ensemble techniques, this method allows insight into the protein folding pathways of HSA, namely intermediate conformations that are accessed by HSA during folding and unfolding.

Using fluorescence correlation spectroscopy to investigate the unfolding of human serum albumin

245th National Meeting (2013)

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