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

Abstract

3 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 environment and having the highest quantum yield of the natural amino acids, however changes in tryptophan florescence 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 only effective when the imidazole sidechain 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 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 on designed peptides and globular proteins. MSe is easily incorporated into proteins, provides a conservative replacement for hydrophobic sidechains, and MSe quenching of Trp fluorescence is pH independent. The oxidized form of MSe, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.

Ultra‐Strong Regenerated Wool Keratin Fibers Regulating via Keratin Conformational Transition

“…Fluorescence quenching is a powerful technique in the field of researching the dynamic changes of keratin structure in complex systems. [ 61–63 ] The remarkable fluorescence quenching effect in fluorescence spectra further supports the adsorption of keratin onto HQSGr (Figure S8B, Supporting Information). In summary, the HQSGr could serve as an ideal reinforcement phase to build the solid and compact interaction with keratin fibrils.…”

77Se NMR Probes the Protein Environment of Selenomethionine

“…The selenium atom is interacting with the indole ring of Trp43. Such favorable electrostatic interactions between selenium and aromatic rings are well documented in proteins. , The selenium is approaching Trp43 edge-on at a distance of 4.75 (A) and 4.72 Å (B) at an angle of 65.3 (A) and 66.8° (B). (The distance was measured from the selenium atom to the center of the indole’s benzene ring; the angle is between the selenium−aromatic ring center line and the normal of the aromatic ring plane; see details in Figure S6.)…”

Maximizing Kinetic Information Gain of Markov State Models for Optimal Design of Spectroscopy Experiments

“…For example, recently, Watson et al have proposed the use of selenomethionine ( M Se ) for Trp quenching to probe protein dynamics. This technique requires a VDW interaction between the Se atom and the Trp residue for quenching to occur via electron transfer . Instead of a mask of 7 Å for Trp–Tyr quenching, a value of 3–4 Å can be used to ensure that the residues will be in the VDW contact range.…”

“…This technique requires a VDW interaction between the Se atom and the Trp residue for quenching to occur via electron transfer. 71 mask of 7 Å for Trp−Tyr quenching, a value of 3−4 Å can be used to ensure that the residues will be in the VDW contact range. Thus, optimal residue pairs can be chosen for experiments employing the M Se quenching of Trp fluorescence.…”