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Synthetic biology of protein folding.

Research paper by Luis L Moroder, Nediljko N Budisa

Indexed on: 15 Apr '10Published on: 15 Apr '10Published in: ChemPhysChem



Abstract

In protein evolution amino acid replacements occur more frequently between similar than dissimilar amino acids. Accordingly, the mutations obey a simple 'similar replaces similar' rule as disruption of the resulting protein structure is minimized by the modest alterations in the amino acid side chains. At laboratory level such non-destructive modifications have to be incorporated in a controlled manner by integrating the chemical diversity achieved in synthetic chemistry into proteins. For this purpose the most straightforward route is generation of the synthetic proteins by the cellular translational apparatus via insertion of isosteric synthetic amino acid analogs during gene expression. This leads to target proteins with chemical diversity not found in nature. Such genetic code engineering does not require any DNA mutagenesis step as it produces extensive sequence variations at the level of protein translation. It is generally achieved either by expansion of the existing amino acid repertoire or by introduction of novel coding units. Here we highlight the concept of using isosteric noncanonical amino acid analogs for in vivo protein synthesis as a useful tool to dissect, study and manipulate protein folding and conformational stability with two examples, the prion protein and green fluorescent protein. In the first example, we show how alternative translation of the single gene sequence by different synthetic amino acids enables the protein to fold into stable, but differing conformations. In particular, replacement of methionine by the isosteric analogs norleucine/metoxinine provide a chemical model to dissect the role of hydrophilicity/hydrophobicity in the alpha-->beta conversion of the prion protein structure. In the second example, proline residues in green fluorescent protein are replaced with (4S)-fluoroproline which improves folding and overall stability of the protein. Indeed, fluorination of the protein matrix creates a network of favorable local interactions absent in the parent (i.e. natural) protein. The generation of these novel properties is the first demonstration of a structural preogranisation principle in one complex globular protein molecule.