Synergy in protein engineering. Mutagenic manipulation of protein structure to simplify semisynthesis.
Semisynthesis is a chemical technique of protein engineering that provides a valuable complement to directed mutagenesis. It is the method of choice when the structural modification requires, for example, a noncoded amino acid. The process involves specific and limited protein fragmentation, structural manipulation of the target sequence, and subsequent religation of fragments to give the mutant holoprotein. We suggested and demonstrated that mutagenesis and semisynthesis could be used synergistically to achieve protein engineering goals otherwise unobtainable, if mutagenesis was used to shuffle methionine residues in the yeast cytochrome c sequence (Wallace, C. J. A., Guillemette, J. G., Hibiya, Y., and Smith, M. (1991) J. Biol. Chem. 266, 21355-21357). These residues can not only be sites of specific cleavage by CNBr but also of spontaneous peptide bond synthesis between fragments in noncovalent complexes, which greatly facilitates the semisynthetic process. We have now used an informed "methionine scan" of the protein sequence to discover other useful sites and to characterize the factors that promote this extraordinary and convenient autocatalytic religation. Of eight sites canvassed, in a wide range of settings, five efficiently provoked peptide bond synthesis. The principal factor determining efficiency seems to be the hydropathy of the religation site. The mutants created have also provided some new insights on structure-function relationships in the cytochrome.
Woods
A C
AC
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
Guillemette
J G
JG
Parrish
J C
JC
Smith
M
M
Wallace
C J
CJ
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D007532:Isoleucine; D007930:Leucine; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D014633:Valine
D007532:Isoleucine; D011392:Proline; D007930:Leucine; D014633:Valine; D008239:Lysine