Watson, MD;
Peran, I;
Zou, J;
Bilse, O;
Raleigh, DP;
(2017)
Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure.
Biochemistry
, 56
(8)
pp. 1085-1094.
10.1021/acs.biochem.6b01000.
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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.
| Type: | Article |
|---|---|
| Title: | Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1021/acs.biochem.6b01000 |
| Publisher version: | http://doi.org/10.1021/acs.biochem.6b01000 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10053602 |
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