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Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure

Author:
Watson, Matthew D., Peran, Ivan, Zou, Junjie, Bilsel, Osman, Raleigh, Daniel P.
Source:
Biochemistry 2017 v.56 no.8 pp. 1085-1094
ISSN:
1520-4995
Subject:
fluorescence, fluorescence emission spectroscopy, fluorescent dyes, histidine, hydrophobicity, pH, peptides, protein folding, proteins, selenomethionine, tryptophan
Abstract:
Fluorescence spectroscopy, relying on intrinsic protein fluorophores, is one of the most widely used methods for studying protein folding, protein–ligand interactions, and protein dynamics. Tryptophan is usually the fluorophore of choice, given its sensitivity to its environment and having the highest quantum yield of the natural amino acids; however, changes in tryptophan fluorescence can be difficult to interpret in terms of specific structural changes. The introduction of quenchers of tryptophan fluorescence can provide information about specific structures, particularly if quenching is short-range; however, the most commonly employed quencher is histidine, and it is effective only when the imidazole side chain is protonated, thus limiting the pH range over which this approach can be employed. In addition, histidine is not always a conservative substitution and is likely to be destabilizing if inserted into the hydrophobic core of proteins. Here we illustrate the use of a Trp-selenomethionine (MSₑ) pair as a specific probe of protein structure. MSₑ requires a close approach to Trp to quench its fluorescence, and this effect can be exploited to design specific probes of α-helix and β-sheet formation. The approach is illustrated using equilibrium and time-resolved fluorescence measurements of designed peptides and globular proteins. MSₑ is easily incorporated into proteins and provides a conservative replacement for hydrophobic side chains, and MSₑ quenching of Trp fluorescence is pH-independent. The oxidized form of MSₑ, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.
Agid:
5635815