Folding of ribonuclease HI from Escherichia coli populates a kinetic intermediate detectable by stopped-flow circular dichroism. Pulse labelling hydrogen exchange reveals that this intermediate consists of a structured core region of the protein, namely helices A and D and beta-strand 4. This kinetic intermediate resembles both the acid molten globule of ribonuclease HI and rarely populated, partially unfolded forms detected under native conditions. These results indicate that the first portion of ribonuclease HI to fold is the most thermodynamically stable region of the native state, and that folding of this protein follows a hierarchical process.

The hydrophobic interaction, the tendency for nonpolar molecules to aggregate in solution, is a major driving force in biology. In a direct approach to the physical basis of the hydrophobic effect, nanosecond molecular dynamics simulations were performed on increasing numbers of hydrocarbon solute molecules in waterfilled boxes of different sizes. The intermittent formation of solute clusters gives a free energy that is proportional to the loss in exposed molecular surface area with a constant of proportionality of 45 ؎ 6 cal͞mol⅐Å 2 . The molecular surface area is the envelope of the solute cluster that is impenetrable by solvent and is somewhat smaller than the more traditional solvent-accessible surface area, which is the area transcribed by the radius of a solvent molecule rolled over the surface of the cluster. When we apply a factor relating molecular surface area to solvent-accessible surface area, we obtain 24 cal͞mol⅐Å 2 . Ours is the first direct calculation, to our knowledge, of the hydrophobic interaction from molecular dynamics simulations; the excellent qualitative and quantitative agreement with experiment proves that simple van der Waals interactions and atomic point-charge electrostatics account for the most important driving force in biology.T he hydrophobic effect is the most important force in stabilizing biological structures ranging from native conformations of proteins to cellular membranes. The origin of this effect has been the topic of much investigation, both experimental and theoretical. Solvent transfer experiments show that for a wide range of different hydrocarbon molecules, the hydrophobic interaction energy depends linearly on the burial of solvent accessible surface area with a constant of proportionality around 25 cal͞mol⅐Å 2 (1-4). Data from calorimetric studies indicate that around room temperature, the hydrophobic effect is primarily entropy driven (5). Both of these results indicate that the interaction must be short-range and must depend on a shell of water molecules. The entropy effect is attributed to the solute imparting additional structure to the surrounding shell waters and reducing their entropy relative to the bulk solvent (6). Unfortunately, these macroscopic studies do little to shed light on our understanding of the detailed microscopic solute-solvent interactions that drive the hydrophobic effect.Although the microscopic details cannot be distinguished by experiment, theoretical studies are ideal for modeling atomiclevel interactions, and a great deal of our understanding of the hydrophobic effect has come from theoretical studies. Classically, there are two aspects to the hydrophobic effect. The first is termed hydrophobic hydration and concerns the effects of the solute on the surrounding water molecules. The second aspect is often referred to as the hydrophobic interaction, the tendency for nonpolar molecules to associate in water. This second aspect is the focus of the present study.A common theoretical treatment of the hydrophobic interaction has been to s...

The origins of the hydrophobic effect are widely thought to lie in structural changes of the water molecules surrounding a nonpolar solute. The spatial distribution functions of the water molecules surrounding benzene and cyclohexane computed previously from molecular dynamics simulations show a high density first hydration shell surrounding both solutes. In addition, benzene showed a strong preference for hydrogen bonding with two water molecules, one to each face of the benzene ring. The position data alone, however, do not describe the majority of orientational changes in the water molecules in the first hydration shells surrounding these solutes. In this paper, we measure the changes in orientation of the water molecules with respect to the solute through spatial orientation functions as well as radial͞angular distribution functions. These data show that the water molecules hydrogen bonded to benzene have a strong orientation preference, whereas those around cyclohexane show a weaker tendency. In addition, the water-water interactions within and between the first two hydration shells were measured as a function of distance and ''best'' hydrogen bonding angle. Water molecules within the first hydration shell have increased hydrogen bonding structure; water molecules interacting across shell 1 and shell 2 have reduced hydrogen bonding structure.hydrophobic effect ͉ orientation I t is widely understood that the structural changes in water induced by exposure to nonpolar surfaces are responsible for the phenomenon known as the hydrophobic effect (1). The small size, tetrahedral geometry, and hydrogen-bonding (H-bonding) ability of water all play a role in the interactions water takes part in with itself as well as with polar and nonpolar solutes. The structure of liquid water is well characterized experimentally in terms of radial distribution functions, g(r), derived from diffraction experiments (2, 3). Similar experiments on solutions of alcohols and tetraalkylammonium ions, however, show very little structural differences in g(r) compared with bulk water (2, 4). Yet the thermodynamics of insertion of nonpolar solutes into water show an unfavorable free energy, a positive entropy at room temperature, and a large positive heat capacity (5, 6). There are several possible reasons why the g(r) may lack sensitivity in detecting structural changes in the hydration shells of hydrophobic solutes: (i) solubility of such solutes is small, so the majority of the observed signal arises from bulk water; (ii) solutes may be incompletely mixed (7); and (iii) the g(r) is inherently insensitive to orientational structure.The tetrahedral geometry of the water molecule and the directional nature of the H-bond impart a highly oriented structure to water. The orientational correlation function of liquid water has been generated by applying maximum entropy methods to g(r) functions (8,9). The resulting density distribution shows clear density peaks beyond the H-bond donor locations (along the O-H bonds), with a broader distribution...

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