A DYNAMIC BUNDLE OF 4 ADJACENT HYDROPHOBIC SEGMENTS IN THE DENATURED STATE OF STAPHYLOCOCCAL NUCLEASE

In an earlier study of the denatured state of staphylococcal nuclease (Wang Y, Shortie D, 1995, Biochemistry 34:15895-15905), we reported ev idence of a three-strand antiparallel beta sheet that persists at high urea concentrations and is stabilized by a local ''non-native'' inter action with four large hydrophobic residues. Because the amide proton resonances for all of the involved residues are severely broadened, th is unusual structure is not amenable to conventional NMR analysis and must be studied by indirect methods. In this report, we present data t hat confirm the important role of interactions involving four hydropho bic residues (Leu 36, Leu 37, Leu 38, and Val 39) in stabilizing the s tructure formed by the chain segments corresponding to beta 1-beta 2-b eta 3-h, interactions that are not present in the native state. Glycin e substitutions for each of these large hydrophobic residues destabili zes or disrupts this beta structure, as assessed by H-N line sharpenin g and changes in the CD spectrum. The C-13 resonances of the carbonyl carbon for several of the residues in this structure indicate conforma tional dynamics that respond in a complex way to addition of urea or c hanges in sequence. Studies of hydrogen exchange kinetics in a closely related variant of staphylococcal nuclease demonstrate the absence of the stable hydrogen bonding between the strands expected for a native -like three-strand beta sheet. Instead, the data are more consistent w ith the three beta strand segments plus the four adjacent hydrophobic residues forming a dynamic, aligned array or bundle held together by h ydrophobic interactions.