PROPERTIES OF THE PROTEIN MATRIX REVEALED BY THE FREE-ENERGY OF CAVITY FORMATION

Background: The classical picture of the hydrophobic stabilization of proteins invokes a resemblance between the protein interior and nonpol ar solvents, but the extent to which this is the case has often been q uestioned. The protein interior is believed to be at least as tightly packed as organic crystals, and was shown to have very low compressibi lity. There is also evidence that these properties are not uniform thr oughout the protein, and conflicting views exist on the nature of side chain packing and on its influence on the properties of the protein. R esults: In order to probe the physical properties of the protein, the free energy associated with the formation of empty cavities has been e valuated for two proteins: barnase and T4 lysozyme. To this end, the l ikelihood of encountering such cavities was computed from room tempera ture molecular dynamics trajectories of these proteins in water. The f ree energy was evaluated in each protein taken as a whole and in submo lecular regions. The computed free energies yielded information on the manner in which empty space is distributed in the system, while the l atter undergoes thermal motion, a property hitherto not analyzed in he terogeneous media such as proteins. Our results showed that the free e nergy of cavity formation is higher in proteins than in both water and hexane, providing direct evidence that the native protein medium diff ers in fundamental ways from the two liquids. Furthermore, although th e packing density was found to be higher in nonpolar regions of the pr otein than in polar ones, the free energy cost of forming atomic size cavities is significantly lower in nonpolar regions, implying that the se regions contain larger chunks of empty space, thereby increasing th e likelihood of containing atomic size packing defects. These larger e mpty spaces occur preferentially where buried hydrophobic sidechains b elonging to secondary structures meet one another. These particular lo cations also appear to be more compressible than other parts of the co re or surface of the protein. Conclusions: The cavity free energy calc ulations described here provide a much more detailed physical picture of the protein matrix than volume and packing calculations. According to this picture, the packing of hydrophobic sidechains is tight in the interior of the protein, but far from uniform. In particular, the pac king is tighter in regions where the backbone forms less regular hydro gen-bonding interactions than at interfaces between secondary structur e elements, where such interactions are fully developed. This may have important implications on the role of sidechain packing in protein fo lding and stability.