Measurement of Submicrosecond Intramolecular Contact Formation in Peptides at the Single-Molecule Level

Abstract

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.

A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate

“…Selective fluorescence quenching of MR121 by Trp arises from photoinduced electron transfer reactions from Trp to the excited fluorophore at van der Waals contact (13,19), a process that is much faster than the temporal resolution of our FCS setup (6.25 ns). Dynamic quenching of fluorophores by electron donors or acceptors results in a decrease in the measured fluorescence lifetime (20), but quenching of MR121 by Trp is dominated by the formation of nonfluorescent complexes where electron transfer occurs on femtosecond to picosecond time scales (21).…”

“…These interaction kinetics are mediated by peptide random-chain diffusion and the manifold intramolecular interaction possibilities of the fluorophore attached to the peptide. Amino acid contact formation kinetics in unstructured peptides are thought to represent elementary steps in protein folding and have been investigated extensively by using various spectroscopic techniques (19,(23)(24)(25).…”

“…Because the amino-terminal fragment itself (TC*F1) shows random-coil behavior, collapse is assisted by nonnative tertiary interactions with the proline-rich carboxyl-terminal domain (residues [12][13][14][15][16][17][18][19][20]. The observed intermediate is characterized by solvent exposure of Trp, which is evidenced by fluorescence quenching of the attached fluorophore within this state, a process that requires van der Waals contact with the Trp side chain (13).…”

Coordinate-dependent diffusion in protein folding

“…A more direct method of obtaining diffusion coefficients for folding is to probe the dynamics of polypeptides or unfolded proteins, for example by using ultrafast spectroscopy to measure rates of contact formation between specific residues (12,(20)(21)(22)(23)(24)(25)(26), or FRETcross-correlation functions to probe the reconfiguration time in the unfolded state (27,28). Determination of the diffusion coefficient in this class of experiments requires that the end-toend distance distribution be known.…”

Hidden Markov Model Analysis of Multichromophore Photobleaching

“…In another example, the fluorescence of an oxazine dye was transiently interrupted by contact with a quencher attached to the other end of a peptide such that the intermittency itself provided the source of the dynamical information. 17 Analysis of intermittency has also been used to learn about the electronic states of semiconductor quantum dots. 18 The full potential of single-molecule experiments is realized when a state-to-state trajectory can be reconstructed from the data.…”