HYDROGEN-BONDING IN PROTEINS AS STUDIED BY AMIDE HYDROGEN D/H-FRACTIONATION FACTORS - APPLICATION TO STAPHYLOCOCCAL NUCLEASE

The D/H fractionation factor (phi) is the extent to which a hydrogen at a particular site becomes enriched in H-2 over H-1 relative to the solvent. A growing body of experimental evidence suggests that there is a correlation between the value of the fractionation factor and hydrogen-bond strength, with a lower phi value reflecting a stronger hydrogen bond. Fractionation factors of 60% of the individual backbone amide hydrogens in the staphylococcal nuclease V8 variant (H124L) have been measured for the enzyme in the presence and absence of bound ligands (the activating ion Ca2+ and the inhibitor thymidine 3',5'-bisphosphate). The method used employed two-dimensional H-1-N-15 nuclear magnetic resonance analysis of uniformly N-15-labeled protein in mixed H2O/D2O solvents. Fractionation factors of individual residues were found to range from 0.3 (T120) to 1.5 (L38). The phi value of 0.3 for the NH of T120, which is the lowest fractionation factor reported for any system yet studied, suggests that the hydrogen bond between T120 HN and D77 O(delta1) is unusually strong. The results of previous site-directed mutagenesis experiments [Hinck, A. P. (1993) Ph.D. Thesis, University of Wisconsin-Madison, Madison, WI] support the notion that formation of this hydrogen bond is important to maintain the stability and conformation of the native state. The phi value averaged over all residues was approximately 0.85 for both the unligated and ligated enzymes. Residues in alpha-helices displayed a slightly lower average phi value (0.79), whereas residues with solvent-exposed amide hydrogens exhibited a slightly higher average figure (0.98). These results suggest that the hydrogen-bond network present in protein alpha-helices may make these intramolecular amide-amide hydrogen bonds stronger than those formed between amide groups and H2O. However, this conclusion must be tempered by the fact that several solvent-exposed amide hydrogens had low phi values. No correlation was found between the phi values and the N-O distances of amide hydrogen-bonded residues, as determined from the X-ray structure of unligated staphylococcal nuclease [Hynes, T. R., & Fox, R. O. (1991) Proteins: Struct., Funct., Genet. 10, 92-105]