Resonance energy transfer and ligand binding studies on pH-induced folded states of human serum albumin.
Human serum albumin (HSA) is a very important transporter protein in the circulatory system. It is a multi-domain binding protein, which binds a wide variety of ligands in its multiple binding sites and aids in transport, distribution and metabolism of many endogenous and exogenous ligands. With change in pH, HSA is known to undergo conformational transformation, which is very essential for picking up and releasing them at sites of differing pH inside physiological system. Hence, the characterization of ligand binding to these pH-induced conformers is extremely important. We have explored binding interaction of a ligand protoporphyrin IX (PPIX), which is demonstrated (X-ray crystallography) to reside in domain-IB at the various pH-induced folded states of HSA. The ligand PPIX is found to remain attached to all the HSA conformers which offers an opportunity to use Förster's resonance energy transfer (FRET) between an intrinsic donor fluorophore (Trp214) located in domain-IIA to the acceptor ligand PPIX to characterize the inter-domain separation between IB and IIA. Additionally FRET between an extrinsic fluorophore 2-p-toluidinylnaphthalene-6-sulfonate (TNS) located in domain-IIIA and PPIX is also undertaken to quantify the inter-domain separation between IB and IIIA. Circular dichroism (CD) and dynamic light scattering (DLS) studies have been done in conjunction with picosecond time resolved fluorescence and polarization-gated spectroscopy to determine, respectively, the secondary and tertiary structures of various pH-induced folded states of the protein. Severe structural perturbation including swelling of the protein is observed in the low pH-induced conformer of HSA as evidenced from all the techniques used.
Shaw
Ajay Kumar
AK
Unit for Nano Science and Technology, Department of Chemical, Biological and Macromolecular Sciences, SN Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India.
Pal
Samir Kumar
SK
D016880:Anisotropy; D002942:Circular Dichroism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009282:Naphthalenesulfonates / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011524:Protoporphyrins / Q000032:analysis / Q000378:metabolism*; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000032:analysis / Q000378:metabolism
D008024:Ligands; D009282:Naphthalenesulfonates; D011524:Protoporphyrins; D012709:Serum Albumin; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D014364:Tryptophan; C028025:protoporphyrin IX