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44740-AC4
Protein Engineering with Non-Coded Amino Acids
Protein design, the modification of proteins to alter their properties in a favorable way, is an extremely active field. However the vast majority of work involves substitution of one of the twenty genetically coded amino acids for another. Advances in protein chemistry together have opened the door to protein design with non-coded amino acids. In principle this offers vast possibilities for protein design however very few systematic studies have been reported. Our work to date has focused on two areas. We have demonstrated the use of D-amino acids to rationally increase protein stability by targeting the unfolded state of proteins and we have explored the use of non-coded fluorescence amino acids to follow protein folding and other conformation changes.
In principle protein stability can be increased by decreasing the entropy of the unfolded state. Such an effect can be induced by the substitution of a glycince with any other aminoacid since glycine lacks a side chain and thus is the most flexible residue. Unfortunately numerous attempts to make use of this strategy have failed because glycine often adopts conformation which is not compatible with an L-aminoacid. One possibility is to use D-aminoacids, however there are conflicting reports on the effects of D-aminoacids substitutions. We have completed an extensive study and have shown that the substitution is stabilizing, quantified the increase in stability to be expected and importantly derived a set of rules for when such substitutions will increase stability. Three conditions need to be met. First the glycine should occupy a solvent exposed site in the folded state, secondly no adverse steric clashes should result from the addition of the new methyl group and thirdly there should be no prolines within 4 to 5 residues N-or C-terminal to the glycine in questions. If these condition are meet an increase in stability of -0.75 to -1.0 Kcal per mole is expected. We have validated this approach on a number of proteins.
Our second line of ACS-PRF supported research has centered on the use of p-Cyanophenyalanine (p-cyanoPhe) as a fluorescent probe. An ideal amino acid analog would exhibit a large, easily interpretable change in fluorescence, but represent only a small perturbation on the structure and hydrophobicity of one or more of the twenty genetically encoded residues, allowing for conservative substitution. p-cyanoPhe appears to meet all of these requirements. Its fluorescent quantum yield is very sensitive to interaction with solvent and is increased significantly in H2O relative to its value in organic solvents. Thus p-cyano-Phe fluorescence is an attractive probe of hydrophobic core formation. p-Cyano-Phe fluorescence can be selectively excited even in the presence of Tyr or Trp because of its blue shifted absorption band. p-Cyano-Phe also has the attractive feature that the cyano group, while an H-bond acceptor, can be readily tolerated in the hydrophobic core since it has a polarity between a methylene group and an amide it is also considerably smaller than Trp and is less polar than Tyr making it a more conservative replacement for Phe. We have shown that p-cyanoPhe fluorescence can be used to follow the self assembly of protein aggregates. As a test case we studied amyloid formation by islet amyloid polypeptide (IAPP). Amyloid formation, in vitro, is traditionally followed using fluorescence detected thioflavin binding experiments. The fluorescence of the dye significantly increases upon binding to the amyloid fibril. The assay is simple to execute, however, it does suffer from noticeable drawbacks. First, the exact mechanism for the fluorescence enhancement is not completely understood, hence, it is not completely clear what the dye binding probes. Second, the dye does not bind to pre-fibrillar intermediates and thus cannot be used to follow their formation. A third, extremely important issue involves the study of inhibitors. Some compounds can bind to amyloid fibrils, displacing bound thioflavin-T without inhibiting amyloid formation. In these cases thioflavin-T assays lead to the incorrect conclusion that the compound is an amyloid inhibitor. Fourth, the dye is an extrinsic probe and there is always the risk that the kinetics of assembly could be affected since the assay is conducted by adding the dye to the peptide solution and it binds to the fibrils as they are forming. IAPP is 37 residues in length, contains a disulfide bond linking residues 2 and 7 and has an amidated C-terminus. We replaced Tyr-37 with p-cyanoPhe. The resulting molecule behaves identically to wildtype confirming the conservative nature of the substitution. Importantly the time course of amyloid formation monitored by the p-cyanoPhe group is identical to that determined by traditional methods.
