Neutron structure of subtilisin BPN prime : Effects of chemical environment on hydrogen-bonding geometrics and the pattern of hydrogen-deuterium exchange in secondary structure elements
- Genetech, Inc., San Francisco, CA (USA)
- Brookhaven National Lab., Upton, NY (USA)
- Univ. of California, San Francisco (USA)
The neutron structure of subtilisin BPN{prime} has been refined and analyzed at 2.0-{angstrom} resolution. The structure studied was a mutant variant of subtilisin, Met222 {yields} Gln, and was used because large, uninhibited crystals could be grown, which was not the case for the native molecule. Comparison of the structure with that of the native molecule indicated that the two structures are essentially the same. Using the capability of the neutron method to locate hydrogen and deuterium atoms, the protonation states of the six histidine residues were assigned. The active site histidine, His64, was found to be neutral at the pH of the analysis (pH 6.1). This group has an unexpectedly low pK{sub a} compared to assignments made by other techniques. The altered pK{sub a} of the group could result from electrostatic effects of other molecules in the crystal lattice. The dihedral conformations of a majority of the hydroxyl rotors were assigned. The hydrogen exchange pattern of subtilisin identified the {beta}-sheet and {alpha}-helix secondary structure elements to be the most resistant to exchange. Fifty-five percent of the peptide amide hydrogens were fully exchanged, 15% unexchanged, and 30% partially exchanged. The largest concentration of unexchanged sites was in the seven-stranded parallel {beta}-sheet, in which there were 11 fully protected groups. Little correlation was found between H-bound length and angle and a peptide group's susceptibility toward exchange. Of the five {alpha}-helices the most protected from exchange is the one defined by residues 224-236. The pattern of exchange identifies regions in this helix where the H-bonding regularity is disrupted.
- OSTI ID:
- 5510413
- Journal Information:
- Biochemistry; (United States), Vol. 30:5; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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