MEASUREMENT OF WATER-AMIDE PROTON-EXCHANGE RATES IN THE DENATURED STATE OF STAPHYLOCOCCAL NUCLEASE BY A MAGNETIZATION-TRANSFER TECHNIQUE

The rates of hydrogen exchange were measured in a ''physiological'' de natured state of staphylococcal nuclease using a NMR magnetization tra nsfer experiment suitable for the measurement of exchange rates faster than 0.5 s(-1). The results are compared with predicted exchange rate s (k(ex)) for the random coil state (Bai et al., Proteins 17:75-86, 19 93). No protection factors (= predicted rate/measured rate) larger tha n 2.4 were observed, consistent with other NMR data which strongly sug gest only small amounts of residual secondary structure in this denatu red state. Systematically low protection factors (0.51 +/- 0.23) were found for Asp and Glu residues, while high protection factors were obs erved for Gly (1.60 +/- 0.60), We conclude that the predicted exchange rates (k(ex)) may have an uncertainty of 2- to 3-fold. Thus, for dena tured proteins only protection factors with a Value of 5 or larger can be assigned structural significance. These results also demonstrate t hat multidimensional magnetization transfer MMR techniques are powerfu l tools in this research field due to its ability to measure rapidly e xchanging protons (>05 s(-1)) with high accuracy. (C) 1997 Wiley-Liss, Inc.