Design consideration and probes for fluorescence resonance energy transfer studies.
Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(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 A 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.
Sinev
M
M
Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel.
Landsmann
P
P
Sineva
E
E
Ittah
V
V
Haas
E
E
D000081:Acetamides; D000263:Adenylate Kinase / Q000737:chemistry* / Q000235:genetics; D000409:Alanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry*; D015195:Drug Design; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D007546:Isoquinolines / Q000737:chemistry; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009281:Naphthalenes / Q000138:chemical synthesis / Q000737:chemistry*; D012459:Salicylates / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
C411325:7-(jodoacetamido)coumarin-4-carboxylic acid; D000081:Acetamides; D003374:Coumarins; D005456:Fluorescent Dyes; D007546:Isoquinolines; C411326:N-iodoacetyl-beta-(2-naphthyl)alanine; D009281:Naphthalenes; D012459:Salicylates; D013438:Sulfhydryl Compounds; C093702:lucifer yellow iodoacetamide; C028387:5-iodoacetamidosalicylic acid; D014364:Tryptophan; D000263:Adenylate Kinase; D000409:Alanine