title: Selenomethionine Quenching of Tryptophan Fluorescence Provides a Simple Probe of Protein Structure.
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. MSerequires 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. MSeis easily incorporated into proteins and provides a conservative replacement for hydrophobic side chains, and MSequenching of Trp fluorescence is pH-independent. The oxidized form of MSe, selenomethionine selenoxide, is also an efficient quencher of Trp fluorescence.
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Watson
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.
authors.forename: Ivan
authors.initials: I
authors.lastname: Peran
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.|Laufer Center for Physical and Quantitative Biology, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zou
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School , Worcester, Massachusetts 01605, United States.
authors.forename: Osman
authors.initials: O
authors.lastname: Bilsel
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11794-3400, United States.|Laufer Center for Physical and Quantitative Biology, Stony Brook University , Stony Brook, New York 11794, United States.|Graduate Program in Biochemistry & Structural Biology, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Daniel P
authors.identifier: 0000-0003-3248-7493
authors.initials: DP
authors.lastname: Raleigh
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D010084:Oxidation-Reduction; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D012269:Ribosomal Proteins / Q000737:chemistry*; D012645:Selenomethionine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D012269:Ribosomal Proteins; C049650:ribosomal protein L9; D014364:Tryptophan; D012645:Selenomethionine