A 1H NMR probe for mobility in the reactive center loops of serpins: spin-echo studies of native and modified forms of ovalbumin and alpha 1-proteinase inhibitor.
It has recently been proposed that the expression of inhibitory activity in serine protease inhibitors (serpins) is a function of the mobility of the extended alpha-helical reactive center loop [Stein, P.E., Leslie, A.G.W., Finch, J.T., Turnell, W.G., McLaughlin, P.J., & Carrell, R.W. (1990) Nature 347, 99-102]. We have employed solution 1H NMR methods, including the Carr-Purcell-Meiboom-Gill (CPMG) and Hahn spin-echo pulse sequences, to try to identify such regions by virtue of their anticipated longer T2 relaxation times in two of the best characterized members of the serpin superfamily, ovalbumin and alpha 1-proteinase inhibitor. The CPMG spectra of native ovalbumin reveal the presence of long-lived resonances from the methyl protons of alanine residues and the CH3 protons of leucine or valine residues as well as the acetyl and ring methine protons of the carbohydrate moieties. Following reaction of ovalbumin with subtilisin Carlsberg to generate plakalbumin [where excision from within the reactive center loop homologue of a hexa- or heptapeptide, with sequence (E)-A-G-V-D-A-A, occurs], its CPMG spectrum retained almost all of the originally present long-lived resonances. Concurrent with the retention of these mobile resonances in plakalbumin is the appearance of two additional resonances consistent with the formation of new C and N termini. On the basis of the proposed mobility of the reactive center loop, it had been expected that removal of the alanine-rich hexapeptide would result in loss of some or all of the long-lived alanine methyl resonances.(ABSTRACT TRUNCATED AT 250 WORDS)
Hood
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville 37232.
Gettins
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D011487:Protein Conformation; D015843:Serpins / Q000737:chemistry*; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
D010446:Peptide Fragments; D015843:Serpins; D000515:alpha 1-Antitrypsin; C038141:plakalbumin; D010047:Ovalbumin