The objective of the research outlined is to determine how the choice of different turn sequences affects protein structure and stability. This work will impact upon: (i) the understanding of how natural proteins fold and function; (ii) the ability to engineer variants of natural proteins with altered structures and desired functions; and (iii) the capability to design entirely novel macromolecules with predetermined structures and biomedically important activities In order to insure that the results of this work are generally applicable, a genetic approach will be employed to generate a large collection of different turn sequences in two different structural contexts. These two structural contexts will be represented by the alpha-helical protein, cytochrome b-562 and the beta-sheet protein, plastocyanin.
The specific aims of the research are: 1) To create a library of different sequences at specific turns in each of these two overexpressed model proteins. The members of the library will each contain a different turn sequence; and together they will represent all possible amino acid sequences in place of the wild-type turn sequence. 2) To develop simple color assays in vivo or in crude cell lysates to screen the libraries for correctly-folded proteins. 3) To determine the turn sequences for representative samplings of these libraries. By comparing the sequences with the results of the color screen, the tolerance for different turn sequences will be determined. 4) To purify and characterize proteins in which turns have been replaced by a variety of different amino acid sequences. By comparing the properties of the substituted proteins, it will be possible to decipher the relationship between the sequence of a turn, and its role in dictating protein structure and stability.
| Ybe, J A; Hecht, M H (1994) Periplasmic fractionation of Escherichia coli yields recombinant plastocyanin despite the absence of a signal sequence. Protein Expr Purif 5:317-23 |
| Johnson, B H; Hecht, M H (1994) Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Biotechnology (N Y) 12:1357-60 |
