Protein Engineering Protocols - Mycobacteriology research center

164 Bradley et al.of length, sequences with the P-N-P-P-N-N-P periodicity form proteins withα-helical secondary structure, whereas those with the P-N-P-N-P-N-P periodicity(examined under the same experimental conditions) form β-sheet structures.The key difference between these two libraries of sequences is the binary patterningitself.2. The binary code codons, NAN and NTN, encode six polar amino acids (glutamate,aspartate, lysine, asparagine, glutamine, and histidine), and five nonpolaramino acids (valine, methionine, isoleucine, leucine, and phenylalanine). In additionto these 11 variable amino acids, a variety of other residues can be incorporatedinto the constant regions of the sequences. For example, our recent libraryof 102 residue four-helix bundles contains 17 of the 20 amino acids (18). Onlyalanine, proline, and cysteine were omitted. In natural proteins, alanine occursboth in surface and core positions. Thus, its role in the binary code as polar ornonpolar is somewhat ambiguous. Proline is a special case because its restrictedφ angle makes it useful only in certain well-defined regions of structure. Cysteineshould be used only in designs wherein a disulfide bond or metal binding isplanned.3. By excluding T from the first position of the NAN (polar) codon, tyrosine codonsare avoided. This is desirable because tyrosine is not a completely polar residueand frequently occurs in the hydrophobic cores of natural proteins. Therefore, onlythe most polar residues (histidine, glutamine, asparagine, lysine, aspartate, andglutamate) are incorporated into the designed surface positions.4. Polyacrylamide gel electrophoresis purification of synthetic oligonucleotides isessential. This reduces the likelihood of truncated oligonucleotides being incorporatedinto the library. Although this purification step reduces the quantity of DNA(and potentially the diversity), the quality of the genes and the resulting librariesis enhanced significantly.AcknowledgmentsSupported by grants from the National Institutes of Health (R01-GM62869),the Army Research Office (DAAG55-98-1-0084), the National ScienceFoundation (MRSEC DMR98-09483), and the Defense Advanced ResearchProjects Agency (n00173-01-1-0015).References1. Lim, W. A. and Sauer, R. T. (1989) Alternative packing arrangements in thehydrophobic core of lambda repressor. Nature 339, 31–36.2. Bowie, J. U., Reidhaar-Olson, J. F., Lim, W. A., and Sauer, R. T. (1990) Decipheringthe message in protein sequences: tolerance to amino acid substitutions. Science247, 1306–1310.3. Axe, D. D., Foster, N. W., and Fersht, A. R. (1996) Active barnase variants withcompletely random hydrophobic cores. Proc. Natl. Acad. Sci. USA 93,5590–5594.