Peptides overlapping sequences

We cannot deduce, from the data given, the sequence of the amino acids in parentheses. To put them in order, we must seek peptides from partial hydrolysis that contain overlapping sequences of these amino acids. 

To establish the amino acid sequence unequivocally it is necessary to have peptides with overlapping sequences. This may be accomplished by determining the sequence of fragments obtained from treating a second aliquot of the protein with chymotrypsin. If these fragments are then treated with trypsin as a check, peptides identical to those obtained previously by successive treatment with trypsin and chymotrypsin are obtained. Other proteolytic enzymes, such as pepsin, subtilisin, and papain, with wider specificity than trypsin and chymotrypsin have proved useful in sequencing of some proteins. 

It can be concluded that the bacterial proteinases can be applied to many proplems where extensive enzymatic proteolysis is required. Because of their wide substrate specificity, they are not useful for producing one of the major sets of peptides which are required to provide overlapping sequences within large polypeptides or proteins. On the other hand, where structural studies of small peptides require extensive cleavage at a variety of bonds, the bacterial proteinases are excellent hydrolytic agents. The products of proteinase action are often quite analogous to the products... 

The protein contains only eight amino acids (C. L. Hew, personal communication). Alanine accounted for 60% of its total residues. The first N-terminal sequence of positions 1-28 was determined on a Beckman sequencer and confirmed by the isolation and analysis of overlapping thermolysin- and subtilisin-digested peptides. This sequence is ... 

By comparison of the amino acid sequence of fragments E and G, the terminal fragments A and D can be matched with their respective adjacent peptides, B and C. Alternatively, the order of the peptides B and C can be deduced from the overlapping sequence in F. 

Deduction of peptide sequence from analysis of overlapping sequences of component peptides. 

The peptides obtained by specific chemical or enzymatic cleavage are separated by some type of chromatography. The sequence of each purified peptide is then determined by the Edman method. At this point, the amino acid sequences of segments of the protein are known, but the order of these segments is not yet defined. How can we order the peptides to obtain the primary structure of the original protein 7Te necessary additional infor mation is obtained from overlap peptides (Figure 3.22). A second enzyme is used to split the polypeptide chain at different linkages. For example, chy-motrypsin cleaves preferentially on the carboxyl side of aromatic and some other bulky nonpolar residues (p, 247). Because these chymotryptic peptides overlap two or more tryptic peptides, they can be used to establish the order of the peptides. The entire amino acid sequence of the polypeptide chain is then known. 

Overlapping Peptide Screening. This method is based on a set of short overlapping synthetic peptides that span the entire sequence of the therapeutic protein of choice (Yeung et al., 2004). For example, in order to screen the variable region of both the heavy and light chain of a therapeutic IgG for potential T cell epitopes, at least different 15-mer peptides, overlapping by... 

The nonspecificity of enzymes used in HX-MS, which fortunately for a set of experimental conditions is reproducible, yields a broad distribution of peptides, many with overlapping sequences resulting in very high sequence coverage, frequently exceeding 90%... 

Peptides generated with overlapping sequences help validate data... 

Partial hydrolysis (Section 24.5D) Random cleavage of a polypeptide with dilute acid, resulting in a mily of peptides of varying lengths that can be more easily sequenced than the parent polpqepr-tide. Once each fragment peptide is sequenced, the areas of overlap indicate the sequence of the initial peptide. 

The fragmentation of the protein is performed by different enzymic and/or chemical techniques, at least by two enzymes of different specifity. The arrangement of the obtained peptides in the same order as they occur in the intact protein is accomplished with the aid of overlapping sequences. The principle of this method is illustrated for subtilisin BPN as an example in Fig. 1.11. 

The peptide shown m green was isolated by trypsin catalyzed hydrolysis and has an ammo acid sequence that completes the remaining overlaps... 

The primary structure of a peptide is given by its ammo acid sequence plus any disulfide bonds between two cysteine residues The primary structure is determined by a systematic approach m which the protein is cleaved to smaller fragments even individual ammo acids The smaller fragments are sequenced and the mam sequence deduced by finding regions of overlap among the smaller peptides... 

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