Conformation-directed recombination of enzyme-activated peptide fragments: a simple and efficient means to protein engineering. Its use in the creation of cytochrome c analogues for structure-function studies.
Protein fragments have been activated by the addition of amino acid esters using proteolytic enzymes under conditions where the equilibria are shifted in the direction of synthesis. Because of the natural propensity of large protein fragments to form complexes approximating native conformation, these activated fragments have been induced to recombine by formation of the missing peptide bond. Although the incorporated ester is only weakly activating, the complex, mimicking an enzyme, provides proximity and orientation at the reacting termini, so that coupling yields are high. In other words, the protein catalyzes its own resynthesis. What distinguishes our technique from the rare natural examples of this phenomenon is that it operates at a variety of cleavage sites and with varying chain lengths. There seems to be no particular limitations on the amino acid esters that can be added, and serine proteases with a wide range of specificities can be used. It thus appears that we have a truly general method for the condensation of large fragments in protein synthesis, be they natural or the products of synthetic or genetic methods. This approach has the advantages over conventional methods of great specificity, high efficiency, and mild conditions of use. With our model protein, cytochrome c, we have used this approach to make analogues that illuminate structure-function relations. Both the highly conserved lysine 39 and the functionally invariant threonine 40 have been replaced by a range of substitutions. The results show how crucial these residues are to the structural and functional integrity of the bottom omega-loop of the protein.
Proudfoot
A E
AE
Département de Biochimie Médicale, Université de Genève, Switzerland.
Rose
K
K
Wallace
C J
CJ
D003488:Cyanogen Bromide; D003574:Cytochrome c Group / Q000378:metabolism*; D004355:Drug Stability; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D003574:Cytochrome c Group; D007202:Indicators and Reagents; D010446:Peptide Fragments; D014357:Trypsin; D013820:Thermolysin; D003488:Cyanogen Bromide