Temperature-Dependent Simultaneous Ligand Binding in Human Serum Albumin

Abstract

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.

A Molecular Magnet Confined in the Nanocage of a Globular Protein

“…[23] This further supports the interaction between the entrapped V 15 and Trp. [22] With an increase in temperature, the donor-acceptor distance (R) increases first gradually and then rapidly as the temperature crosses 330 K ( Figure 5), which is very much consistent with the irreversible thermal denaturation of the protein. The increase in R indicates a temperature-induced interdomain separation of the protein, as evidenced earlier by the increase in the size of the protein measured by the dynamic light scattering (DLS) technique.…”

“…This is an initial melting of the globule state that leads to an irreversible unfolding of the protein for temperatures higher than 328 K. [20][21][22] In the temperature range from 293 to 333 K the CD spectrum of HSA suffers only a moderate effect, but when the temperature is increased to 343 K considerable change is produced (Figure 4). Specific changes occurring at the fluorophore-containing region of the protein can be probed by specific FDCD spectra of the protein.…”

“…The increase in R indicates a temperature-induced interdomain separation of the protein, as evidenced earlier by the increase in the size of the protein measured by the dynamic light scattering (DLS) technique. [21,22] In this way, V 15 is applied as a molecular marker for the protein folding process.…”

Protein Binding: Do We Ever Learn?

“…As the impact of temperature on PB of drugs is well described, all models investigating any aspect of PB of antimicrobials should be performed at physiological temperature (25,92).…”

Assessment of the Dissociation Energetics of Some Selected Ligand Drugs Bound on Human Serum Albumin by Differential Scanning Calorimetry

“…Most of the aforementioned techniques are conducted isothermally whereas most binding interactions are temperature dependent (12). In addition to these techniques, calorimetry may very well be suitable to detect the binding phenomena (13).…”