Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

Abstract

The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

Probing of the rates of alternating access in LacY with Trp fluorescence

“…This process may be much slower than the rate of closure on the cytoplasmic side that brings the ends of helices V and X into close proximity. As demonstrated by phosphorescence studies (47), His quenching of Trp occurs over a very short range and is highly dependent upon orientation, which dramatically decreases quenching rates in proteins.…”

Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

“…Structural Fluidity and Homogeneity as Derived from the Phosphorescence Lifetime, , in Buffer at 273 K-Another sensitive parameter of the local protein/solvent mobility is the intrinsic phosphorescence lifetime, which decreases from about 6 s in rigid matrices to (sub) milliseconds in fluid solutions (11). Time-resolved measurements provide also information on the structural homogeneity of the protein site, as stable states of the protein ensemble differing in local flexibility will exhibit distinct lifetimes resulting in multiexponential phosphorescence decays.…”

“…The observation of multiple P values for a single Trp position reflects the presence of different protein conformational states, which do not rapidly interchange on the time scale of P . The ability to quench the Trp triplet state is governed by the local viscosity (), and the relation between P and is well established (10,11). The information obtained by measuring P , combined with the recording of emission spectra of the protein in both the glass-state and in the fluid state, provides site-specific, structural information about the Trp microviscosity (), micropolarity, and protein conformational heterogeneity.…”

Tertiary Contact Formation in α-Synuclein Probed by Electron Transfer

“…The second-order rate constant for the bimolecular ET quenching reaction is near the diffusion limit (7 × 10 9 M -1 s -1 ; 20 mM sodium phosphate buffer, pH 7.4). 18 The relatively long-lived 3 W* excited state (τ ) 40-60 µs) is produced upon 290-nm laser excitation and monitored by transient absorption spectroscopy using an Ar-ion laser probe (457.9 nm). The quenching kinetics show conclusively that the protein is highly dynamic and that conformers are interchanging rapidly on the microsecond time scale (Figure 1).…”