44740-AC4
Protein Engineering with Non-Coded Amino Acids
Our ACS supported research was focused on the application of non genetically coded aminoacids in protein design. The vast majority of the work in protein design involves substitution of one of the twenty genetically coded amino acids for another. Advances in peptide synthesis as well as the development of expressed protein ligation methodology and the realization of the so called 21st pair methodology (which allows the recombinant incorporation of certain non-coded aminoacids) have provided exciting new possibilities for the use of non-natural aminoacids in protein design, however very few systematic studies have been reported. Our ACS-PRF supported work was focused on the use of D-amino acids to rationally increase protein stability by targeting the unfolded state of proteins and on the use of non-coded fluorescence amino acids to follow protein folding and other conformation changes.
Topic-1 “Mirror Image Protein Design” Protein stability can be increased by decreasing the entropy of the unfolded state. Unfolded state entropy can be decreased by the substitution of a glycine with any other aminoacid since glycine lacks a side chain and thus is the most conformationally flexible residue in terms of the number of allowed backbone conformations. Numerous attempts to make use of this strategy have failed because glycine often adopts a conformation which is not compatible with an L-aminoacid, for example at the C-termini of helices or in certain turns. One possibility to overcome this issue is to use D-aminoacids since they have a Ramachadran plot which is a mirror image of the one associated with L-aminoacids. There were conflicting reports on the effects of D-aminoacids substitutions before we started our work. We have shown that the substitution is generally stabilizing, quantified the increase in stability to be expected and 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 question. If these conditions are met we expect an increase in stability of -0.70 to -1.0 Kcalmol-1. We used the villin headpiece as a test system for some of these studies and as part of our work determined the structure of the domain.
Topic-2 “Intrinsic and Selective Fluorescence Probes to Follow Amyloid Formation and Protein Folding” Our second line of research was centered on the application of p-cyanophenyalanine (Fcn) as a fluorescent probe. Fcn is a useful fluorescent analog for incorporation into proteins and peptides. It represents a very small perturbation to a Phe or Tyr. Its fluorescent quantum yield is very sensitive to interaction with solvent and is increased significantly in water relative to its value in organic solvents. Fcn fluorescence can be selectively excited in the presence of Tyr or Trp. Fcn has the attractive feature that the cyano group is tolerated in the hydrophobic core of proteins. It is also smaller than Trp and is less polar than Tyr making it a more conservative replacement for Phe. We have showed that Fcn fluorescence can be used to follow protein folding and 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 mechanism of the fluorescence enhancement is not completely understood. Second, the dye does not bind to pre-fibrillar intermediates and thus cannot be used to follow their formation. A third 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 can lead to the incorrect conclusion that the compound is an amyloid inhibitor. The standard assay can give false results because of these effects while our Fcn labeled proteins accurately and faithfully report on amyloid formation. IAPP contains three aromatic residues and we replaced each individually by an Fcn. The resulting molecules behaved identically to wild type confirming the conservative nature of the substitution. Importantly the time course of amyloid formation monitored by the Fcn group was identical to that determined by traditional methods. We also showed that Cl- is an effective quencher of Fcn fluorescence and have used this effect to conduct time resolved studies of the environment of the three side chains during amyloid formation. This study highlights the utility of the approach we developed with the assistance of ACS-PRF funding.
