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Biochemistry. 2003 Mar 18;42(10):2847-56.

NMR determination of pKa values for Asp, Glu, His, and Lys mutants at each variable contiguous enzyme-inhibitor contact position of the turkey ovomucoid third domain.

Author information

1
National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.

Abstract

From the larger set of 191 variants at all the variable contact positions in the turkey ovomucoid third domain, we selected a subset that consists of Asp, Glu, His, and Lys residues at eight of the nine contiguous P6-P3' positions (residues 13-21), the exception being P3-Cys16 which is involved in a conserved disulfide bridge. Two-dimensional [1H,1H]-TOCSY data were collected for each variant as a function of sample pH. This allowed for the evaluation of 31 of the 32 pK(a) values for these residues, the exception being that of P5-Lys14, whose signals at high pH could not be resolved from those of other Lys residues in the molecule. Only two of the titrating residues are present in the wild-type protein (P6-Lys13 and P1'-Glu19); hence, these measurements complement earlier measurements by A. D. Robertson and co-workers. This data set was supplemented with results from the pH dependence of NMR spectra of four additional single mutants, P1-Leu18Gly, P1-Leu18Ala, P2-Thr17Val, and P3'-Arg21Ala, and two double mutants, P2-Thr17Val/P3'-Arg21Ala and P8-Tyr11Phe/P6-Lys13Asp. Probably the most striking result was observation of a P2-Thr17...P1'-Glu19 hydrogen bond and a P1'-Glu19-P3'-Arg21 electrostatic interaction within the triad of P2, P1', and P3' (residues 17, 19, and 21, respectively). In several cases, the pK(a) of a particular residue was sensed by resonances not only in that residue but also in residue(s) with which it interacts. Remarkably, in several interacting systems, resonances from different protons within the same residue yielded different pHmid values.

PMID:
12627950
DOI:
10.1021/bi0269512
[Indexed for MEDLINE]

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