title: Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide.
abstract: Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent (N,N',N'-trimethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N'-(p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.
authors.affiliation: Department of Chemistry, University of Waterloo, Waterloo, Ont., Canada N2L 3G1.
authors.forename: Shenhui
authors.initials: S
authors.lastname: Lang
authors.forename: Donald E
authors.initials: DE
authors.lastname: Spratt
authors.forename: J Guy
authors.initials: JG
authors.lastname: Guillemette
authors.forename: Michael
authors.initials: M
authors.lastname: Palmer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D001593:Benzyl Compounds / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry; D007202:Indicators and Reagents; D008715:Methionine / Q000737:chemistry*; D015335:Molecular Probes / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012645:Selenomethionine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D001593:Benzyl Compounds; C039092:CAMP protein, Streptococcus; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D007202:Indicators and Reagents; D015335:Molecular Probes; D011506:Proteins; D012645:Selenomethionine; D008715:Methionine; D003545:Cysteine; C038682:benzyl bromide