C-peptide of ribonuclease

Osterhout JJ Jr, Baldwin RL, York EJ, Stewart JM, Dyson HJ, Wright PE (1989) 11 NMR studies of the solution conformations of an analogue of the C-peptide of ribonuclease A. Biochemistry 28 7059-7064. 

Simulated annealing has also been applied to the conformational study of dipeptide models of Gly, Ala, and Asp pentaglycine and Leu-enkephalin [11] an analog of vasopressin [10] (3S, 4S)-statin [12] analogs of thyrotropin releasing hormone [13] and the C-peptide of Ribonuclease A [13]. 

D.Y. Jackson, J. Burnier, C. Quan, M. Stanley, J. Tom, J.A. Wells, A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues. Science 266(5381) (1994) 243-247. 

Two other types of specific side-chain interactions have been proposed to stabilize the a helix formed by C-peptide (Shoemaker et al., 1987b). A salt bridge between Glu-2 and Arg-10 has been detected in the crystal structure of ribonuclease A (Wlodawer and Sjolin, 1983) as well as in C-peptide in aqueous solution (Rico et al., 1986). This salt bridge also fixes the N-terminal boundary of the helix between Glu-2 and Thr-3. It is not sterically possible to make this ion pair if Glu-2 is part of the helix. Synthetic studies in which either Glu-2 or Arg-10 is replaced by Ala have provided support for the importance of this interaction in stabilizing the a-... 

The exploitation of ribonuclease (66,71,72) in depleting nucleic acids involves the incubation of the extracted protein (containing the cellular RNA) around neutral pH at 55°C for one or two hours to activate the RNase and hydrolyze the nucleic acids. Unfortunately, these conditions also result in the activation of the proteolytic enzymes of yeast cells. These concurrently hydrolyze the proteins to low molecular weight peptides and significantly reduce the yield of intact proteins. 

Schultz observed (Schultz et al., 1961) that on partial hydrolysis of a commercial preparation of ribonuclease with 0.03 N HCl for times varying up to 48 hr at 105°C, up to 14 out of the 15 bound aspartic residues were liberated. He recommends the method for rapid comparative fingerprinting of the resulting peptides from proteins such as human and animal sera, heme proteins, pepsin, and albumins. 

Ribonuclease S, of knowm crystal structure (10), presents an instructive case. Except in the region of residues 18-23, it is very similar to RNase A (11). CjNj modifies known salt-bridge pairs (5). The distance between the C s of ALA20 and SER 21 is 27 A calculated from the X-ray diffraction based coordinates (12), however, considerable uncertainty exists relative to structural parameters for residues 18-23 (12). Are residues 20 and 21 transiently associated and susceptible to C2N2 driven condensation The answer appears to be yes but not as a reestablished peptide bond. Instead, it appears to be a depsi-pepMe ester link. 

Ijeucine aminopeptidase has been applied in many ways to particular problems in structural analysis of peptides and proteins. Sequences in the amino-terminal portion of a peptide can often be established by measurement of the order of appearance of amino acids that are released during hydrolysis. The procedure has been used with a variety of proteins and peptides, including ribonuclease (Hirs et al., 1960), hemoglobin (Konigsberg and Hill, 1962, 1963 Schroeder et al., 1963), cytochrome c (Margoliash, 1962 Matsubara and Smith, 1963), and lysozyme (Canfield, 1963). 

This system is then tested with a standard protein mixture containing 0.1 pmol of ribonuclease A, cytochrome c, myoglobin, insulin and p-lactoglobulin, 20 pmol of carbonic anhydrase II and bovine serum albumin and 1 pmol of CCK flanking peptide. The proteins and peptide are focused by CIEF and then divided into seven pi fractions by stop and go operation. Figure 5.5 shows the RPLC-MS analysis of these fractions. The labels A-G... 

Prepn. 3, 9 (1953). Bovine pancreatic RNase A is a single-chain peptide of 124 amino add residues. Primary structure C. H. W. Hits et al J. Biol. Chem. 235, 633 (I960). Amino acid sequence D. H. Spackman et al.. Ibid. 648. Pictorial representation of entire structure Stein, Moore, Sci. Am. 204, no. 2, 81-92 (Feb. 1961). Ribonuclease from plant leaves has slightly different characteristics Markham, Strominger, Biochem. J. 64, 46p (1956). Can be obtained as a by-product of microbial erythromydn production Japan, pat. 263>38( 63) (to Shionogi). Chemical synthesis of materials possessing partial RNase enzyme activity Gutte,... 

In the early sixties Ernesto Scoffone, (Plate 40) professor at the University of Padua, with his associates, F. Marchiori and R. Rocchi, embarked on a study of active esters and soon employed them for the synthesis of biologically important molecules such as the S-peptide. This 20-residue fragment of ribonuclease A is cleaved by subtilisin from the N-terminal part of the chain of the enzyme. The paduan school soon became a significant center of peptide research where a number of S-peptide analogs have been prepared and utilized in investigations of the ribonuclease S system. Several of Scoffone s coworkers, G. Borin, A. Fontana, C. Di Bello, L. Moroder, E. Peggion, A. Scatturin, A.M. Tamburro, C. Toniolo and G. Vidali turned into frequent contributors of the peptide field. 

As an example. Figure 3 shows some backbone-backbone time correlation functions computed from a 1 ns solvated simulation of an analogue of the ribonuclease C-peptide. ... 

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