J. Ferrie, Rebecca F. Wissner, E. Petersson
Jan 27, 2015
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Journal
Biophysical Journal
Abstract
Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that is known to stabilize protein structure. Previous studies have shown that the addition of TMAO can induce folding of thermodynamically unstable proteins, causing them to regain high functional activity. In solution, monomeric α-synuclein (αS) is intrinsically disordered. Our laboratory and others have shown that αS undergoes significant compaction in the presence of TMAO. Previously, we have demonstrated that p-cyanophenylalanine and a thioamide can serve as a minimally perturbing probe pair for Forster Resonance Energy Transfer (FRET) experiments. Despite the utility of this pair in measuring short intramolecular distances, inclusion of the thioamide is synthetically intensive, rendering it difficult to generate a large library of double-labeled mutants for FRET studies. As an alternative, we have expressed a library of double-labeled αS mutants containing the genetically encodable FRET pair, Cnf and tryptophan (Trp). This set of double-labeled proteins will allow us to obtain a more comprehensive description of the TMAO-induced morphology of αS.