Conformational
Fluctuation Dynamics of Domain I of
Human Serum Albumin in the Course of Chemically and Thermally Induced
Unfolding Using Fluorescence Correlation Spectroscopy
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Abstract
The
present study elucidates the involvement of conformational
fluctuation dynamics during chemically and thermally induced unfolding
of human serum albumin (HSA) by fluorescence correlation spectroscopic
(FCS) study, time-resolved fluorescence measurements, and circular
dichroism (CD) spectroscopic methods. Two fluorescent probes, tetramethylrhodamine-5-maleimide
(TMR) and <i>N</i>-(7-dimethylamino-4-methylcoumarin-3-yl)
iodoacetamide (DACIA) were used to selectively label the domain I
of HSA through the reaction with cys-34 for these studies. The guanidine
hydrochloride (GnHCl) induced global structural change of HSA is monitored
through its hydrodynamic radius (<i>r</i><sub>H</sub>) and
CD response, which is found to be two step in nature. In FCS experiment,
along with the diffusion time component we have observed an exponential
relaxation time component (τ<sub>R</sub>) that has been ascribed
to the concerted chain dynamics of HSA. Unlike in the global structural
change, we found that the τ<sub>R</sub> value changes in a different
manner in the course of the unfolding. The dependence of τ<sub>R</sub> on the concentration of GnHCl was best fitted with a four
state model, indicating the involvement of two intermediate states
during the unfolding process, which were not observed through the
CD response and <i>r</i><sub>H</sub> data. The fluorescence
lifetime measurement also supports our observation of intermediate
states during the unfolding of HSA. However, no such intermediate
states were observed during thermally induced unfolding of HSA