The interaction of bovine serum albumin (BSA) with gold colloids and surfaces was studied using zeta-potential and quartz crystal microbalance (QCM) measurements, respectively, to determine the surface charge and coverage. The combination of these two measurements suggests that BSA binding to gold nanoparticles and gold surfaces occurs by an electrostatic mechanism when citrate is present. The binding of BSA to bare gold is nearly two times greater than the binding of BSA to a citrate-coated gold surface, suggesting that protein spreading (denaturation) on the surface may occur followed by secondary protein binding. On the other hand, binding to citrate-coated gold surfaces can be fit to a Langmuir isotherm model to obtain a maximum surface coverage of (3.7 +/- 0.2) x 10(12) molecules/cm(2) and a binding constant of 1.0 +/- 0.3 microM(-1). The zeta-potential measurements show that the stabilization of colloids by BSA has a significant contribution from a steric mechanism because the colloids are stable, even at their isoelectric point (pI approximately 4.6). To be consistent with the observed phenomena, the electrostatic interactions between BSA and citrate must consist of salt-bridges, for example, of the carboxylate-ammonium type, between the citrate and the lysine on the protein surface. The data support the role of strong electrostatic binding but do not exclude contributions from steric or hydrophobic interactions with the surface adlayer.

Equilibrium Fourier transform infrared (FTIR) and temperaturejump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 s at 60.2°C) and double phenylalanine mutant (3.01 s at 49.9°C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 s at 49.9°C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 s compared with 6.97 s for the WT at 49.9°C.protein folding ͉ Fourier transform IR ͉ temperature jump ͉ diffusion collision T he mechanism by which an unfolded polypeptide chain folds into its final native structure is still not fully understood, despite the obvious importance of the protein folding problem. In recent years there has been considerable interest in small single-domain proteins that fold quickly. These proteins provide attractive systems for computational and theoretical studies, and they are useful experimental models of the early steps in the assembly of more complicated folds. The helical subdomain derived from the villin headpiece, HP36, is a small three-helix structure found in the extreme C terminus of villin ( Fig. 1). Its modest size and helical structure suggest that it should fold rapidly, and this hypothesis has been independently confirmed by fluorescence-detected temperature-jump (T-jump) studies and NMR lineshape analysis (1-3). The helical subdomain is probably the smallest occurring natural sequence that has been shown to fold cooperatively. Its small size and very rapid folding have made it the focus of a large number of theoretical and computational studies (4-18).Residual structure and interactions in the unfolded state may play an important role in determining the rate of protein folding. Unfolded state structure might contribute to rapid folding by constraining and limiting the initial conformational search in the unfolded basin. Some models of folding postulate an important role for residual unfolded state structure. The diffusion collision model, for example, has been applied to rapidly folding helical proteins, and a key parameter in the model is the intrinsic stability of the individual microdomains (11,19,20). Although there have been a number of experimental studies of fast folding proteins, surprisingly little work has been reported that ...