Ribonuclease peptide

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... 

Solid phase peptide synthesis does not solve all purification problems however Even if every coupling step m the ribonuclease synthesis proceeded in 99% yield the product would be contaminated with many different peptides containing 123 ammo acids 122 ammo acids and so on Thus Memfield and Gutte s six weeks of synthesis was fol lowed by four months spent m purifying the final product The technique has since been refined to the point that yields at the 99% level and greater are achieved with current instrumentation and thousands of peptides and peptide analogs have been prepared by the solid phase method... 

Thannhauser, T. W., McWherter, C. A., and Scheraga, H. A., Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography. II. A two-dimensional technique for determination of disulfide pairings using a continuous-flow disulfide detection system, Anal. Biochem., 149, 322, 1985. 

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. 

Straub, J.E. Rashkin, A. Thirumalai, D., Dynamics in rugged energy landscapes with applications to the S-peptide and Ribonuclease A, J. Am. Chem. Soc. 1994,116, 2049... 

Simonson, T. Brimger, A. T., Thermodynamics of protein-peptide binding in the ribonuclease S system studied by molecular dynamics and free energy calculations.,... 

This technique has been described as a general method of studying protein-protein interactions as well as a method for investigating the three-dimensional structure of individual proteins (Muller et al., 2001 Back et al., 2003 Dihazi and Sinz, 2003 Sinz, 2003 Sinz, 2006). It also has been used for the study of the interactions of cytochrome C and ribonuclease A (Pearson et al., 2002), to investigate the interaction of calmodulin with a specific peptide binder (Kalkhof et al., 2005a Schmidt et al., 2005), and for probing laminin self-interaction (Kalkhof et al., 2005b). 

Vithayathil, P.J., and Richards, F.M. (1960) Modification of the methionine residue in the peptide component of ribonuclease-S./. Biol. Chem. 235, 2343-2351. 

H Romovacek, SR Dowd, K Kawasaki, N Nishi, K Hofmann. Studies on polypeptides. 54. The synthesis of a peptide corresponding to positions 24-104 of the peptide chain of ribonuclease I, . J Am Chem Soc 101, 6081, 1979. 

F. X. Schmid, R. Grafl, A. Wrba, and J. J. Beintema, Role of proline peptide bond isomerization in unfolding and refolding of ribonuclease, Proc. Natl. Acad Sci. U.S.A. 83, 872-876 (1986). 

Fig. 1. Schematic drawing of the polypeptide backbone of ribonuclease S (bovine pancreatic ribonuclease A cleaved by subtilisin between residues 20 and 21). Spiral ribbons represent a-helices and arrows represent strands of /3 sheet. The S peptide (residues 1-20) runs down across the back of the structure.

Sokolovsky, M., and A. Patchornik Nonenzymatic cleavage of peptide bonds The hall cystine residues in bovine pancreatic ribonuclease. J. Amer. chem. Soc. 86, 1859—1860 (1964). 

Commercially available computer-automated peptide synthesizers allow synthesis of a polypeptide in a time of about one hour or less per amino acid unit. The ribonuclease synthesis takes about one week or less. 

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. 

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