Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes.
The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).
Ahmad
Basir
B
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Ankita
Khan
Rizwan Hasan
RH
D020106:Acrylamide / Q000737:chemistry / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002725:Chloroform / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D007536:Isomerism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D012709:Serum Albumin; D020106:Acrylamide; D014443:Tyrosine; C027132:1-anilino-8-naphthalenesulfonate; D002725:Chloroform; D014508:Urea