Protein sequencing specific peptide bond cleavage

Proteolytic enzymes have been used extensively in the study of protein structui e. First, of course, purified proteases have been employed to catalyze the cleavage of specific peptide bonds in proteins in the process of establishing amino acid sequences and distributions. Within the last few years it has also become apparent that proteolytic enzymes can be utilized to obtain at least semiquantitative information about protein (and nucleic acid) configuration (or secondary-tertiary structure) as well as about amino acid sequence. This use of proteases can be divided into two general areas ... 

Fig. 3. Specificity and sites of cleavage of the clostridial neurotoxins. VAMP is bound to the SSV membrane through a single transmembrane domain (black box), with the majority of the protein exposed to the cytoplasm. In addition, VAMP contains an amino-terminal domain rich in proline (hatched box). SNAP-25 and syn-taxin are bound to the target membrane via palmitoylation (SNAP-25) or via a single transmembrane domain (syntaxin). TeTx and BoNT/B, D, F or G act on the conserved central portion of VAMP and release its amino-terminal part into the cytosol. The sequences indicate the peptide bonds cleaved by CNTs on rat VAMP-1 and VAMP-2. BoNT/A and E cleave SNAP-25 at the carboxyl terminus, with the release of nine and twenty-six residues peptides respectively. BoNT/C also cleaves SNAP-25 at the carboxy-terminus, and cleaves syntaxin at a single site near the cytosolic membrane surface. The action of TeTx and BoNT/B, C, D, F and G causes the release of a large portion of the cytosolic domain of VAMP and syntaxin. Conversely, only a small segment of SNAP-25 is released by the selective proteolysis of BoNT/A, C and E...

Cyanogen bromide (BrC=N) causes the hydrolysis of the amide bond on the C-side of a methionine residue. Cyanogen bromide is more specific than the endopeptidases about what peptide bonds it cleaves, so it provides more reliable information about the primary structure (the sequence of amino acids). Because cyanogen bromide is not a protein and therefore does not recognize the substrate by its shape, cyanogen bromide will still cleave the peptide bond if proline is at the cleavage site. 

Factor XII is a secreted protein and would be expected to contain a signal peptide which functions in transport of the protein across the rough endoplasmic reticulum membrane (76). The cDNA sequence predicts that factor XII is synthesized as a precursor containing an amino-terminal extension of at least 19 amino acid residues. Cleavage of a Ser-Ile bond in the precursor would give rise to plasma factor XII. This bond cleavage is consistent with the specificity of signal peptidase (77). 

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