Temperature-dependent simultaneous ligand binding in human serum albumin.
Human serum albumin (HSA) is a soluble protein in our circulatory system, which is known to bind a variety of drugs and ligands. Since Sudlow's pioneering works on the ligand-binding sites, a major effort of the biophysical/biochemical research has been directed to characterize the structural, functional, and dynamical properties of this protein. Structural studies on HSA have revealed distinct temperature-induced folded states. Despite knowing about the ligand-binding properties and residues important for the binding, less is understood about the temperature-dependent molecular recognition of the protein. Here, we have prepared thermally induced unfolded states of the protein and characterized those by circular dichroism (CD) and differential thermal analysis (DTA) techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering (DLS) measurements. We have used two fluorescent ligands (4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran) (DCM; hydrophobic; neutral) and Nile blue (NB; cationic) of different natures to characterize the ligand-binding properties of the protein in the native and thermally unfolded states. The possible binding sites of the ligands have been characterized by competitive binding with other drug molecules having definite binding sites in HSA. Picosecond-resolved Förster resonance energy transfer (FRET) studies along with steady-state and polarization-gated spectroscopies on the ligands in the protein reveal the dynamics of the binding sites at various temperatures. From the FRET studies, an attempt has been made to characterize the simultaneous binding of the two ligands in various temperature-dependent folded states of HSA.
Sinha
Sudarson Sekhar
SS
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.
Mitra
Rajib Kumar
RK
Pal
Samir Kumar
SK
D002942:Circular Dichroism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D008024:Ligands; D012709:Serum Albumin; D012997:Solvents