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Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use 19F nuclear magnetic resonance spectroscopy to follow the reversible, two‐state unfolding thermodynamics of the N‐terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein–crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG–protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG–protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered. [ABSTRACT FROM AUTHOR]

Ubiquitous in the cellular milieu, small organic compounds termed osmolytes help to regulate the response to environmental stress. Elasmobranchii, such as sharks, accumulate high concentrations of several osmolytes, most notably urea, trimethylamine N‐oxide (TMAO), and glycine betaine (GB). These three compounds are used to osmoregulate the organism's body fluids, so that they counteract seawater salinity, as well as the destabilizing effects of hydrostatic pressure on cells and their molecular components. Herein we focus on glycine betaine and show how it modifies interactions between lipid membranes. We find that the addition of GB exerts an apparent attractive force that draws neighboring membranes toward one another. We show that, at the molecular level, this apparent attraction between membranes in the presence of GB is due to its preferential exclusion from the space between adjacent membranes, which thereby exerts an osmotic pressure that brings membranes closer together. This action is similar to the one we have previously reported for TMAO. However, we find that the net effect of GB is significantly smaller than that of TMAO, because GB concomitantly significantly weakens van der Waals attractions between membranes by changing the dielectric properties of the intervening solution. Finally, we show how GB acts in combination with urea. At high total concentrations and over a wide range of GB‐to‐urea ratios, urea counteracts the effect of GB, so that the equilibrium separation between membranes is close to their values in pure water. This finding supports the prevailing idea that mixtures of several osmolytes can achieve minimal net impact on biomacromolecular stability while also counteracting osmotic stress. [ABSTRACT FROM AUTHOR]

Electrofreezing experiments of super‐cooled water (SCW) with different ions, performed directly on the charged hemihedral faces of pyroelectric LiTaO3 and AgI crystals, in the presence and in the absence of pyroelectric charge are reported. It is demonstrated that bicarbonate (HCO3−) ions elevate the icing temperature near the positively charged faces. In contrast, the hydronium (H3O+) slightly reduces the icing temperature. Molecular dynamics simulations suggest that the hydrated trigonal planar HCO3− ions self‐assemble with water molecules near the surface of the AgI crystal as clusters of slightly different configuration from those of the ice‐like hexagons. These clusters, however, have a tendency to serve as embryonic nuclei for ice crystallization. Consequently, we predicted and experimentally confirmed that the trigonal planar ions of NO3− and guanidinium (Gdm+), at appropriate concentrations, elevate the icing temperature near the positive and negative charged surfaces, respectively. On the other hand, the Cl− and SO42− ions of different configurations reduce the icing temperature. [ABSTRACT FROM AUTHOR]

We describe a new approach to calculate the binding of flexible peptides and unfolded proteins to multicomponent lipid membranes. The method is based on the transfer matrix formalism of statistical mechanics recently described as a systematic tool to study DNA-protein-drug binding in gene regulation. Using the energies of interaction of the individual polymer segments with different membrane lipid species and the scaling corrections due to polymer looping, we calculate polymer adsorption characteristics and the degree of sequestration of specific membrane lipids. The method is applied to the effector domain of the MARCKS (myristoylated alanine rich C kinase substrate) protein known to be involved in signal transduction through membrane binding. The calculated binding constants of the MARCKS(151-175) peptide and a series of related peptides to mixed PC/PS/PIP2 membranes are in satisfactory agreement with in vitro experiments. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Driven by conditions set by smaller solutes, proteins fold and unfold. Experimentally, these conditions are stated as intensive variables - pH and other chemical potentials - as though small solutes were infinite resources that come at an externally varied free energy cost. Computationally, the finite spaces of simulation allow only fixed numbers of these solutes. By combining the analytic Gibbs adsorption isotherm with the computational Monte Carlo sampling of polymer configurations, we have been able to overcome an inherent limitation of computer simulation. The idea is to compute analytically the free energy changes wrought by solutes on each particular configuration. Then numerical computation is needed only to sample the set of configurations as efficiently as when no bathing solute is present. For illustration, the procedure is applied to an idealized two-dimensional heteropolymer to yield lessons about the effect of cosolutes on protein stability. Proteins 2004. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]