Protein Engineering Protocols - Mycobacteriology research center

Combinatorial Protein Design Strategies 15Fig. 2. Amino acid probabilities of the SH3 domain (PDB:1CKA). Calculated profile(❚); sequence alignment-based profile ( ). F10, L18, F20, and L26 are located athydrophobic core sites with fractional solvent accessible surface areas less than 20%.amino acids and rotamer states per site decreases with decreasing energy. Asshown in Fig. 1, C vreaches a valley at approximately T c(= 1/β c) = 2 mol/kcal, afterpassing a peak at approx 10 mol/kcal, at which point, the identities and conformationsof residues in the interior become relatively well defined (59); whereas surfaceresidues, although predominantly hydrophilic, have a large number of rotamerstates of comparable probability. This is consistent with the conformational variabilityof surface-exposed residues among proteins in the structural database.Thus, the heat capacity is helpful in determining at which “effective temperature”the probabilities should be examined. In addition, direct comparisons of the calculatedprofile with those obtained by sequence alignments (e.g., the homologyderivedsecondary structure of proteins database; ref. 66) yields good agreement,especially for buried regions (Fig. 2).2.4. Gene Libraries for Protein ProfilesIn Subheading 1.3., we discussed how to make use of a particular structure’ssequence profile. If a specific sequence is identified, either as a consensussequence or a directed search, this may be straightforwardly realized in anexperiment using peptide synthesis or by expressing a synthetic gene that codesfor the sequence. Larger proteins are most often realized using gene expression.If the probabilistic sequence information is to be used to construct a combinatoriallibrary (see Note 4), methods are needed that transcribe the proteinprofiles into libraries of partially random gene sequences. Nonuniform distributionsof nucleotides are necessary to encode peptide sequences that have