The ileal lipid binding protein (ILBP or I-BABP) binds bile salts with positive cooperativity and has unusual site selectivity, whereby cholic acid binds preferentially in one site and chenodeoxycholic in another, despite both sites having an affinity for both ligands and the ligands only differing by a single hydroxyl group. Previous studies of the human variant have assumed that the ligand/protein binding ratio is 2:1, but we show, using electrospray ionization mass spectroscopy, that human ILBP binds bile acids with a 3:1 ratio, even at low protein and ligand concentrations. Docking calculations and molecular dynamics (MD) simulations identify an allosterically active binding site on the protein exterior that induces a change from a closed conformation to an open one, characterized by a movement of one of the α-helices by ~10° with respect to the β-clam shell. Additional independent MD simulations of several hundred nanoseconds implicate the change between conformations in the mechanisms of both cooperativity and ligand site selectivity.
Staphylococcal nuclease (SNase) is a model protein that contains one domain and no disulfide bonds. Its stability in the native state may be maintained mainly by key amino acids. In this study, two point‐mutated proteins each with a single base substitution [alanine for tryptophan (W140A) and alanine for lysine (K133A)] and two truncated fragment proteins {positions 1–139 [SNase(1–139) or W140O] and positions 1–141 [SNase(1–141) or E142O]} were generated. Differential scanning microcalorimetry in thermal denaturation experiments showed that K133A and E142O have nearly unchanged ΔHcal relative to the wild‐type, whereas W140A and W140O display zero enthalpy change (ΔHcal≈ 0). Far‐UV CD measurements indicate secondary structure in W140A but not W140O, and near‐UV CD measurements indicate no tertiary structure in either W140 mutant. These observations indicate an unusually large contribution of W140 to the stability and structural integrity of SNase.
Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.
Differential scanning calorimetry, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and numerical simulations were used to study the thermostability of the N-terminal RNA-binding domain (RBD) of the SARS-CoV nucleocapsid protein. The transition temperature of the RBD in a mixing buffer, composed of glycine, sodium acetate, and sodium phosphate with 100 mM sodium chloride, at pH 6.8, determined by differential scanning calorimetry and circular dichroism, is 48.74 degrees C. Experimental results showed that the thermal-induced unfolding-folding transition of the RBD follows a two-state model with a reversibility >90%. Using a simple Gō-like model and Langevin dynamics we have shown that, in agreement with our experiments, the folding of the RBD is two-state. Theoretical estimates of thermodynamic quantities are in reasonable agreement with the experiments. Folding and thermal unfolding pathways of the RBD also were experimentally and numerically studied in detail. It was shown that the strand beta(1) from the N-terminal folds last and unfolds first, while the remaining beta-strands fold/unfold cooperatively.
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