1,720,965 research outputs found
Chemical cleavage of tryptophanyl and tyrosyl peptide bonds via oxidative halogenation mediated by o-iodosobenzoic acid.
No full text
Probing the Molten Globule State of alpha-Lactalbumin by Limited Proteolysis
No full textLimited proteolysis has been used to probe the partially folded state of bovine alpha-lactalbumin (BLA) at acid pH (A-state) or dissolved in aqueous trifluoroethanol (TFE-state). The sites of proteolytic fission have been determined by isolation of the various BLA fragments and comparison of their N-terminal amino acid sequence and amino acid composition after acid hydrolysis, as well as their molecular mass determined by mass spectrometry, with the known sequence of BLA. Incubation of BLA with pepsin at 20-22 degrees C and pH 2.0 in the presence of 0.1 M NaCl results in very rapid cleavage of the 123-residue chain at peptide bond Ala40-Ile41 and subsequently at Leu52-Phe53, leading to a nicked species of BLA constituted by the two fragments 1-40 and 53-123 cross-linked by the four disulfide bridges of the protein. Much slower proteolytic cleavage occurs at Tyr103-Trp104. The highly helical conformational state acquired by BLA when dissolved in aqueous buffer (pH 7.0) containing 50% (v/v) TFE was probed by the TFE-resistant thermolysin. Proteolytic cleavage occurs at the peptide bond Ala40-Ile41 and much more slowly at Phe80-Leu81. Moreover, the peptide bond Gln2-Leu3 at the N-terminus of the chain is partially cleaved by thermolysin. Conversely, native BLA in a pH 7.0 buffer is rather resistant to proteolysis
Limited Proteolysis as a Tool to Detect Structure and Dynamic Features of Globular Proteins: Studies on Thermolysin
No full textAutolysis of thermolysin. Isolation and characterization of a folded three-fragment complex.
No full textIncubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis
Correlation between sites of limited proteolysis and segmental mobility in thermolysin.
No full textLimited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes
- …
