Thiol ester cleavage-dependent conformational change in human alpha 2-macroglobulin. Influence of attacking nucleophile and of Cys949 modification.
Cleavage of the thiol ester that exists between the side chains of Cys949 and Gln952 in human alpha 2-macroglobulin (alpha 2M) destabilizes the native conformation and leads to a large-scale conformational change that results in exposure of the receptor binding domain and to changes in electrophoretic mobility and sedimentation coefficient. The basis of this destabilization of the alpha 2M native conformation following thiol ester cleavage is not understood. We have extended observations that chemical modifications of the newly-formed SH in thiol ester-cleaved alpha 2M can slow the rate of conformational change in an attempt to determine the factors that influence the kinetic stability of the native conformation. Using changes in the fluorescence of alpha 2M-bound 6-(p-toluidino)-2-naphthalenesulfonic acid, we have determined the rate constant for conformational change in human alpha 2M, following thiol ester cleavage by ammonia, methylamine, or ethylamine, both in the absence and in the presence of an SH-modifying group, methyl methanethiosulfate. The influence of bait region cleavage in half of the alpha 2M tetramer on this rate has been examined by comparing the properties of native alpha 2M with those of I-form alpha 2M. The properties of two recombinant alpha 2M variants, C929S and C949Q, have also been examined. We found that the stabilizing effects of Cys949 and Gln952 modification were synergistic and optimal for S-thiomethylation in conjunction with methylamine cleavage of the thiol ester. Modification of Gln952 in the absence of SH modification was destabilizing.(ABSTRACT TRUNCATED AT 250 WORDS)
Gettins
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Department of Biochemistry, University of Illinois-Chicago 60612-4316, USA.
D000641:Ammonia / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004952:Esters; D005021:Ethylamines / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004952:Esters; D005021:Ethylamines; D005456:Fluorescent Dyes; D008744:Methylamines; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D000641:Ammonia; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; C027451:methylamine; D003545:Cysteine; C041564:ethylamine