Ribonuclease peptide sequences

S-protein a cleavage product of ribonuclease, representing amino acid residues 21-124 of the ribonuclease primary sequence. It is produced, together with S-peptide (positions 1-20), by the action of sub-tilisin on ribonudease. 

Cysteine-Containing Peptide Sequences in Ovalbumin and Ribonuclease... 

Memfield successfully automated all the steps m solid phase peptide synthesis and computer controlled equipment is now commercially available to perform this synthesis Using an early version of his peptide synthesizer m collaboration with coworker Bemd Gutte Memfield reported the synthesis of the enzyme ribonuclease m 1969 It took them only SIX weeks to perform the 369 reactions and 11 391 steps necessary to assemble the sequence of 124 ammo acids of ribonuclease... 

An example of a noncovalent system has already been discussed in the case of ribonuclease A (see Section 5.1.6). 29 32 Thus, much information was gained from the relatively easy synthesis of many peptide analogues with sequences derived from either end of the 124-residue protein, and their combination with the protein segments RNase(21-124), RNase(l-118), or RNase(21-118) produced by selective enzymatic digestions. 

Ribonuclease A was the first enzyme to be synthesized in the laboratory. Fully active ribonuclease has been synthesized,752 as have new modified enzymes. For example a 63-residue peptide made up of five segments of the native RNase sequence retained measurable catalytic activity.753 Using total synthesis, unnatural amino acids, such as 4-fluorohistidine, have been incorporated at specific positions in RNAse.752... 

A bacterial peptidase splits a 20-residue fragment containing His 12 from the N-terminal end of RNase A. This "S-peptide" can be recombined with the rest of the molecule, which is inactive, to give a functional enzyme called ribonuclease S. In a similar way, residues 119-124 of RNase A can be removed by digestion with carboxypeptidase to give an inactive protein which lacks His 119. In this case, a synthetic peptide with the sequence of residues 111 -124 of RNase A forms a complex with the shortened enzyme restoring full activity.755... 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

In the synthesis of the enzyme ribonuclease by the Merrifield method, the 124 amino acids were arranged in the ribonuclease sequence through 369 reactions and some 12,000 individual operations of the automated peptide-synthesis machine without isolation of any intermediates. 

Identify the signal peptide and family signature sequences of pig pancreatic ribonuclease with the given amino acid sequence ... 

The hard-sphere potential has also been used to compute the sterically allowed conformations of small polypeptides of known amino acid sequence, viz. an octapeptide loop of ribonuclease (Nemethy and Scheraga, 1965) and the cyclic decapeptide gramicidin-S (Vanderkooi et al., 1966). We shall use the calculation for gramicidin-S to illustrate the method. This decapeptide consists of two identical pentapeptides joined into a ring by peptide bonds, the amino acid sequence being... 

Over 50% of the aspartic acid of the proteins of hepatic origin is liberated in 16 hours, but the gamma-globulins of extrahepatic origin required 36 hours to reach 50%. The extent to which this reflects certain characteristics of the primary structure is described in similar studies on insulin, ribonuclease, and glucagon (37). The cleavage of the peptide chain at the aspartic acid bonds may release other amino acids when they are between aspartic acid residues, when the aspartic acid is penultimate at either end of the chain, or if the other residues occupy positions of particularly labile sequences not known at this time. The... 

Below is part of the structure of ribonuclease surrounding one of the catalytic amino acids Hisl2. There are seven amino acids in this sequence. Every one is different and every one has a functionalized side chain. This is part of a run of ten amino acids between Phe8 and Alai 9. This strip of peptide has six different functional groups (two acids, one each of amide, guanidine, imidazole, sulfide, and alcohol) available for chemical reactions. Only the histidine is actually used. 

This process is routinely automated in commercially available machines. Solutions of all of the protected amino acids required are stored in separate containers and a programmed sequence of coupling and deprotection leads rapidly to the complete peptide in days rather than the years needed for solution chemistry. The most dramatic illustration of this came with the publication of a heroic traditional synthesis of bovine pancreatic ribonuclease A (an enzyme with 124 amino acids) by Hirschmann, side-by-side with one by Merrifield using functionalized polystyrene as we have described. The traditional method required 22 co-workers, while the Merrifield method needed only one. 

Certain indications for a selectivity of bonding are obtained by a comparison of the occurrence of peptide bonds of the types AB and BA in some individual proteins (Sorm et al, 1961). In a sufficiently long peptide chain the statistical probability of both these combinations should, of comse, be equal in the majority of cases, indeed, the experimental data are in agreement with statistical expectation, but in some instances there are deviations, as in the structure of ribonuclease, or in the a- and (3-chains of hemoglobin where there are differences in the incidence of several dipeptide combinations of the two sequences senses (Table III). 

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