Conformational fluctuation dynamics of domain I of human serum albumin in the course of chemically and thermally induced unfolding using fluorescence correlation spectroscopy.
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 N-(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 (r(H)) 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(R)) that has been ascribed to the concerted chain dynamics of HSA. Unlike in the global structural change, we found that the τ(R) value changes in a different manner in the course of the unfolding. The dependence of τ(R) 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 r(H) 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.
Department of Chemistry Indian Institute of Technology Kanpur , Kanpur 208 016, UP, India.
Rajeev
R
Yadav
Bhaswati
B
Sengupta
Pratik
P
Sen
D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D057446:Hydrodynamics; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D012235:Rhodamines / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D005456:Fluorescent Dyes; D012235:Rhodamines; D012709:Serum Albumin; C005358:tetramethylrhodamine; D019791:Guanidine