Proximity of thiol esters and bait region in human alpha 2-macroglobulin: paramagnetic mapping.
The two key structural features of alpha 2-macroglobulin (alpha 2M) involved in inhibitory caging of proteases are the thiol ester and the bait region. This paper examines the environment of the hydrolyzed thiol ester in methylamine-treated human alpha 2M and the separation between the bait region and the thiol ester and between the four thiol esters in the tetramer to try to further our understanding of how bait region proteolysis triggers thiol ester cleavage. The sulfhydryl groups of Cys-949, formed upon cleavage of the thiol ester by methylamine, were specifically labeled with the nitroxide spin-labels 3-(2-iodoacetamido)-PROXYL (iodo-I) (PROXYL = 2,2,5,5-tetramethylpyrrolidine-1-oxyl), 3-[2-(2-iodoacetamido)acetamido]-PROXYL (iodo-II), and 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (iodo-III). ESR spectra of these alpha 2M derivatives showed that label I is firmly held and label II has limited freedom of rotation consistent with location of the cysteine residue in a narrow cavity. Label III has much greater motional freedom. From the absence of dipole-dipole splittings in the ESR spectra, it is concluded that the four nitroxide groups in the tetramer are more than 20 A apart for both label I and label II. Label I broadens 1H NMR signals from one phenylalanyl, one tyrosyl, and four histidyl residues in the bait region. Separations of 11-17 A are estimated between the nitroxide of label I and these residues. Label II is further away and only broadens resonances from one of the histidines.(ABSTRACT TRUNCATED AT 250 WORDS)
Gettins
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Beth
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Cunningham
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D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D046911:Macromolecular Substances; D011487:Protein Conformation; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins / Q000302:isolation & purification
D046911:Macromolecular Substances; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins