Design consideration and probes for fluorescence resonance energy transfer studies

Michael Sinev, Pavel Landsmann, Elena Sineva, Varda Ittah, Elisha Haas

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-β-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 Å in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General Considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.

Original languageEnglish (US)
Pages (from-to)352-362
Number of pages11
JournalBioconjugate Chemistry
Volume11
Issue number3
DOIs
StatePublished - May 2000

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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