Protein sequencing disulfide bond position

A prior distribution for sequence profiles can be derived from mixtures of Dirichlet distributions [16,51-54]. The idea is simple Each position in a multiple alignment represents one of a limited number of possible distributions that reflect the important physical forces that determine protein structure and function. In certain core positions, we expect to get a distribution restricted to Val, He, Met, and Leu. Other core positions may include these amino acids plus the large hydrophobic aromatic amino acids Phe and Trp. There will also be positions that are completely conserved, including catalytic residues (often Lys, GIu, Asp, Arg, Ser, and other polar amino acids) and Gly and Pro residues that are important in achieving certain backbone conformations in coil regions. Cys residues that form disulfide bonds or coordinate metal ions are also usually well conserved. 

The partial amino acid sequence of Gla-containing proteins from calf bone and swordfish bone shows that 21 of the 45 residues common to each are identical. The three Gla-residues and the single disulfide bond are in identical relative positions in the two proteins. An important difference between both proteins is the absence of 4-hydroxyproline in the swordfish protein638. The kidney has been localized as the site of vitamin K-dependent Gla-protein biosynthesis637. Gla has been reported to be a constituent of fossil bones639. ... 

The linear sequence of amino acids joined together by peptide bonds is termed the primary structure of the protein. The position of covalent disulfide bonds between cysteine residues is also included in the primary structure. 

Each PIR entry consists of Entry (entry ID), Title, Alternate names, Organism, Date, Accession (accession number), Reference, Function (description of protein function), Comment (e.g., enzyme specificity and reaction, etc.), Classification (superfamily), Keywords (e.g., dimer, alcohol metabolism, metalloprotein, etc.), Feature (lists of sequence positions for disulfide bonds, active site and binding site amino acid residues, etc.), Summary (number of amino acids and the molecular weight), and Sequence (in PIR format, Chapter 4). In addition, links to PDB, KEGG, BRENDA, WIT, alignments, and iProClass are provided. 

Sequences of the representative proteins are displayed in JOY protein sequence/ structure representation (http //www-cryst.bioc.cam.ac.uk/ joy/). The representations are uppercase for solvent inaccessible, lowercase for solvent accessible, red for a helix, blue for strand, maroon for 310 helix, bold for hydrogen bond to main chain amide, underline for hydrogen bond to main chain carbonyl, cedilla for disulfide bond, and italic for positive angle. The query sequence is displayed in all capital letters. The consensus secondary structure (a for a helix, b for strand, and 3 for 310 helix) as defined, if greater than 70% of the residues in a given position in that particular conformation, is given underneath. 

If certain amino acids (such as disulfide bonds or crucial amino acids in the hydrophobic core) are indispensable for protein stability, these positions can be changed by site-directed mutagenesis (Proba et al., 1998). To avoid back-mutations during the evolution process or the selection of a residual wild-type contamination, the pool is amplified after each round of ribosome display with a primer that reintroduces the destabilizing mutation. If the mutation is not close to one of the termini, the coding sequence has to be amplified in two parts, which are then reassembled by PCR. Thus, to evolve improved stabilities this strategy first removes known crucial stabilizing factors to select for compensatory mutations at different positions. 

The system of intraglobular residue-residue contacts of a protein of N residues may be represented as an N x N matrix of the carbon-alphas, whose elements are ones (contact) or zeros (lack of contact). Any reasonable definition of contact provides ones in the positions (i, i + l)that correspond to a peptide bond between two adjacent residues in the sequence. The same is true for the residues corresponding to the pair of cysteines forming a disulfide bond (these data may not be available as input and may be used as a test of correct prediction). This set of contacts describes the sequential covalent topology and is a constant part of the contact matrix which does not depend on the spatial structure of the polypeptide chain however, any additional information on existing intraglobular contacts (e.g., from NMR data or disulfide linkage) can easily be introduced in the constant part of the contact matrix A ... 

Fig. 17.7. Engineered protein designed for mechanochemistry studies, (a) A pair of cysteine residues introduced into the 127 protein (positions 32 and 75 suifur atoms as spheres) spontaneously form a buried disuifide bond. (b). In response to an unfolding force, the protein extends right up to the disuifide bond. Unfoiding exposes the disulfide bond to the solution, (c) Then, a nucieophiie such as DTT can initiate a Sn2 reaction, leading to the reduction of the disuifide bond and the concomitant extension of the amino acids that were trapped behind the disuifide bond. This sequence of events nnambiguousiy identifies individual disulfide bond reduction events, allowing for the stndy of a pulling force on a Sn2 chemicai reaction...

There are 25 cysteines in apoB 16 of these are known to be involved in disulfide bonds, 7 are known to be free, and the remaining two, Cys-2906 and Cys-4326, are unknown (Yang et al., 1990). Only two of the free cysteines of apoB, Cys-3734 and Cys-4190, are labeled in LDL by the fluorescent probe 5-iodoacetamidofluorescein, possibly because they are exposed on the surface of the lipoprotein (Coleman et al., 1990). Of the 8 disulfide bonds in apoB 100, 7 are within 1000 amino acids of the N terminus of the protein (6 in the first 500 amino acids). The first two disulfides, Cys -Cys and Cys -Cys °, cross the remainder of the disulfides are between nearest neighbors. The number of amino acids separating the cysteines in each disulfide is relatively small, 5-49 amino acids the exception is Cys -Cys , wherein the cysteines are separated by 130 amino acids. This disulfide is apparently unique to the human sequence, because cysteines are not found in homologous positions in rat, mouse, Syrian hamster, pig, rabbit, or chicken apoB (Law and Scott, 1990). The disulfide pattern of apoB 100 is shown schematically in Fig. 1C. 

Fig. 5. Platform sequence alignments of the MHC class I-like ligands of NKG2D. Sequences of MIC-A and -B, the ULBPs and the RAE-ls have been aligned, divided by family and domain, using CLUSTALW (Thompson et al, 1994). Note that the alignments across families are only very approximate at these levels of sequence identity. Sequences have been numbered from the initiator methionine in the leader peptide, but only the residues in the mature proteins have been shown. Cysteines have been highlighted, and disulfide bond partners have been indicated with matching symbols (, f). For the MIC sequences, allelic substitutions have been indicated by the additional residues shown below the sequences (deletions are indicated with an X ). Diamonds below the sequences indicate NKC2D contact positions, based on the known complex structures (MIC-A 001, ULBPS, and RAE-1/5).

Chemokines, a sub-family of the cytokines. They are composed of a core domain containing two or three disulfide bonds in a flexible N-terminal domain whose truncation affects the potency of receptor activation. The chemokines are homologous 8- to 10-kDa proteins with 20 to 70% sequence homology. They are subdivided into at least four families based on the relative position of the Cys residues in the mature protein, although only the a- and -chemokines (both of which contain four Cys residues) have been well characterized. In the a-chemokines (CXC chemokines), the first two Cys residues are separated by a single amino acid residue (Cys-Xaa-Cys), whereas in the -chemokines (CC chemokines) the first two Cys residues are adjacent to each other (Cys-Cys). a-Chemokines are roughly 70 to 130 aa in size and are secreted with leader sequences of 20-25 aa which are cleaved before release. Besides the conserved Cys-Xaa-Cys motif near the N-terminus of the protein. 

In the native state, all the proteins contain a half-cystine residue as the carboxyterminal amino acid, and most have the aminoterminal blocked with an acetylalanine (there are a few exceptions in the ITT A family). The components of IIIA and B2 families exhibit a characteristically cystine-rich repetitive pentapeptide (-cys-cys-X-pro-Y-), which is not found in the IIIB family. The X position of the pentapeptide is frequently occupied by glutamine, glutamic acid, or arginine residues, while serine or threonine usually occurs in the Y position. The above sequence pattern has been found to occupy the major portion of the B2 protein [25,224]. In this protein, an intrachain disulfide bond is thought to link cys-2 of the first pentapeptide to cys-1 of the second pentapeptide unit, which would result in a convoluted pattern in the protein backbone [30,31,148,232]. 

Comparative Peptide Mapping This procedure is used when the protein is cleaved with specific cleaving agents and the sequence of the protein is known. A simple molecular mass measurement of the peptides in a protein digest will identify the position of a disulfide bond in the protein. 

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