Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level.
We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.
Physikalisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany.
Hannes
H
Neuweiler
Andreas
A
Schulz
Martin
M
Böhmer
Jörg
J
Enderlein
Markus
M
Sauer
D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D010078:Oxazines / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism
D005456:Fluorescent Dyes; D010078:Oxazines; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D010649:Phenylalanine; D014364:Tryptophan