Triplet State Of Tryptophan Research Articles

Photoinduced Electron Transfer from the Tryptophan Triplet State in Zn-Azurin.

Tryptophan is one of few residues that participates in biological electron transfer reactions. Upon substitution of the native Cu2+ center with Zn2+ in the blue-copper protein azurin, a long-lived tryptophan neutral radical can be photogenerated. We report the following quantum yield values for Zn-substituted azurin in the presence of the electron acceptor Cu(II)-azurin: formation of the tryptophan neutral radical (Φrad), electron transfer (ΦET), fluorescence (Φfluo), and phosphorescence (Φphos), as well as the efficiency of proton transfer of the cation radical (ΦPT). Increasing the concentration of the electron acceptor increased Φrad and ΦET values and decreased Φphos without affecting Φfluo. At all concentrations of the acceptor, the value of ΦPT was nearly unity. These observations indicate that the phosphorescent triplet state is the parent state of electron transfer and that nearly all electron transfer events lead to proton loss. Similar results regarding the parent state were obtained with a different electron acceptor, [Co(NH3)5Cl]2+; however, Stern-Volmer graphs revealed that [Co(NH3)5Cl]2+ was a more effective phosphorescence quencher (K SV = 230 000 M-1) compared to Cu(II)-azurin (K SV = 88 000 M-1). Competition experiments in the presence of both [Co(NH3)5Cl]2+ and Cu(II)-azurin suggested that [Co(NH3)5Cl]2+ is the preferred electron acceptor. Implications of these results in terms of quenching mechanisms are discussed.

Direct excitation of tryptophan phosphorescence. A new method for triplet states investigation

We studied room temperature phosphorescence of tryptophan (TRP) embedded in poly (vinyl alcohol) films. With UV (285 nm) excitation, the phosphorescence spectrum of tryptophan appears at about 460 nm. We also observed the TRP phosphorescence with blue light excitation at 410 nm, well outside of the S0 →S1 absorption. This excitation reaches the triplet state of tryptophan directly without the involvement of the singlet excited state. The phosphorescence lifetime of tryptophan is in the sub-millisecond range. The long-wavelength direct excitation to the triplet state results in high phosphorescence anisotropy which can be useful in macromolecule dynamics study via time-resolved phosphorescence.

Solvent Relaxation Significantly Contributes to Electron Transfer Rates between Tryptophan Triplet State and Cystine

Understanding how electron transfer (ET) is affected by the dynamics of the medium is of fundamental significance for energy production in biology. ET is also used in many experimental techniques. A recent example is photo-induced electron transfer (PET), which is being employed to measure the rates of intra-molecular contact formation in intrinsically disordered proteins (IDPs) and polymers. Such measurements reveal important information on the structure and dynamics of IDPs. In this setting, tryptophan and cysteine are particularly useful probes, as they can be mutated into a protein sequence with minimal perturbation.

1P062 トリプトファン三重項寿命測定を用いたstaphylococcal nucleaseの変性構造の評価(01C. 蛋白質 : 物性,ポスター,第52回日本生物物理学会年会(2014年度))

1P062 トリプトファン三重項寿命測定を用いたstaphylococcal nucleaseの変性構造の評価(01C. 蛋白質 : 物性,ポスター,第52回日本生物物理学会年会(2014年度))

Folding Kinetics of Small Proteins Revealed by Tryptophan-Cysteine Contact Formation Experiments

Spectroscopic probes sensitive to the formation intra-chain contacts are increasingly used to study the structural and dynamical properties of polypeptides. Quenching of the triplet state of tryptophan by close contact with cysteine enables the measure of contact formation rates without the need of extrinsic probes, thus being suitable for the study of natural proteins and peptides. We illustrate the use of this method to investigate the conformational dynamics of small two-state proteins and of beta-hairpin peptides in conditions close to native. The coexistence of folded and unfolded states can be revealed from the non-exponential relaxation of the excited triplet, enabling the characterization of both unfolded chain dynamics and folding kinetics. The comparison with protein fragments and model disordered peptides allows estimating the contribution of different chain regions to the folding process. Using this approach, we outline the kinetic pathway leading to the formation of the beta-hairpin structure of the C-terminal fragment of protein GB1, revealing the presence of misfolded states, as proposed in recent computational studies.

Measuring Sub-Microsceond Protein Folding Kinetics with Independent Probes

Determining the rate of forming the truly folded conformation of ultrafast folding proteins has been an important issue for both experiment and simulation. The double-norleucine mutant of the 35-residue villin subdomain has been the focus of recent computer simulations with atomistic molecular dynamics because it is currently the fastest folding protein, with folding times less than 1 microsecond as measured by tryptophan fluorescence in laser temperature jump experiments. However, the molecular dynamics simulations reported folding times much longer than the times for fluorescence changes. To establish the folding time with greater certainty, we have therefore employed an independent method. In this experiment the decay of the tryptophan triplet state measured by triplet-triplet absorption spectroscopy monitors the change in the population of the unfolded state. The experimental rates from the two methods will be critically compared with each other and with the rates from simulations.T.C. and M.B. made equal contributions to this work.

Conformations and Dynamics of Polypeptide Chains Revealed By Tryptophan-Cysteine Contact Formation Kinetics

Spectroscopic probes sensitive to intra-chain contact formation events in polypeptides are increasingly used to study the conformational and dynamical properties of different amino acid sequences. Quenching of the triplet state of tryptophan by close contact with cysteine enables the measure of contact formation rates without the need of extrinsic probes, thus being suitable for the study of natural proteins and peptides. We illustrate the use of this method to investigate the unfolded state of small proteins in conditions close to native and the kinetics of weakly structured protein fragments. The coexistence of different conformational states can be revealed from the non-exponential relaxation of the excited triplet, enabling the characterization of both the chain dynamics for each state and the transition kinetics. Moreover, the rate of contact formation measured for the least structured states is compared with those observed for model disordered peptides, allowing to estimate the strength of electrostatic and hydrophobic interactions between residues other than the probes. We test this approach with the widely studied GB1 15-residue C-terminal, which folds into a beta-hairpin structure. The kinetics of elementary conformational steps leading to the folded state is outlined, revealing the presence of misfolded states as proposed in recent computational works.

Phosphorescence from Single Tryptophan in Amorphous Solid Human Serum Albumin Exhibits Solvent-Protein Dynamics Slaving

The physical properties of amorphous biomolecules are important to the texture and stability of low-moisture foods, the stability of pharmaceuticals, the permeability of edible films, and the viability of organisms during anhydrobiosis. Protein stability is often improved via the inclusion of small-molecule excipients during freeze-drying and organisms overproduce sugars such as sucrose or trehalose during anhydrobiosis. The effect on internal protein dynamics caused by substitution of a protein's surface water molecules with small sugar molecules is unclear. To explore this question, we have analyzed tryptophan phosphorescence decays of human serum albumin (HSA) in the dry amorphous solid state. Phosphorescence is an ideal approach, as the long-lived triplet state of tryptophan is sensitive to the long time-scale molecular motions of proteins in the dry state. Human serum albumin (HSA) was chosen because it contains a single, buried tryptophan residue and thus can provide information on the local dynamics of a specific site in the interior of the protein. Amorphous protein films were prepared by spreading concentrated solutions of HSA with and without sugar onto quartz slides, followed by rapid drying and extensive desiccation. Phosphorescence intensity decays were collected and fit with multiple exponential functions. From the average lifetime of these fits the rates of non-radiative decay (kNR) of the triplet state were calculated; kNR is dependent on the microviscosity of the site and is thus a measure of molecular mobility of the HSA tryptophan site. At all temperatures this measure of molecular mobility was lower in the films containing sucrose. Break-point analysis of a kNR Arrhenius plot revealed two temperature regimes with a transition occurring at the glass transition temperature of sucrose. Research supported in part by the National Research Initiative of USDA-CSREES.

Conformational Properties of Unfolded HypF-N

We have measured the intramolecular diffusion rate between distant residues in the aggregation-prone protein HypF-N under various denaturing conditions. Using the method of cysteine quenching of the tryptophan triplet state, we find that intramolecular diffusion remains roughly constant at high concentrations of denaturant (2-6 M GdnHCl) and slows down at low concentrations of denaturant, but the decrease is not uniform throughout the chain. Extrapolation of these measurements to 0 M GdnHCl gives D approximately 10(-7) cm(2) s(-1), about 1 order of magnitude lower than unstructured peptides and at least 2 orders of magnitude higher than well-behaved proteins. This suggests that there is a dynamic range of conformational reorganization within which partially unfolded states are prone to aggregation.

Kinetics of Contact Formation and End-to-End Distance Distributions of Swollen Disordered Peptides

Unstructured polypeptide chains are subject to various degrees of swelling or compaction depending on the combination of solvent condition and amino acid sequence. Highly denatured proteins generally behave like random-coils with excluded volume repulsion, whereas in aqueous buffer more compact conformations have been observed for the low-populated unfolded state of globular proteins as well as for naturally disordered sequences. To quantitatively account for the different mechanisms inducing the swelling of polypeptides, we have examined three 14-residues peptides in aqueous buffer and in denaturant solutions, including the well characterized AGQ repeat as a reference and two variants, in which we have successively introduced charged side chains and removed the glycines. Quenching of the triplet state of tryptophan by close contact with cysteine has been used in conjunction with Förster resonance energy transfer to study the equilibrium and kinetic properties of the peptide chains. The experiments enable accessing end-to-end root mean-square distance, probability of end-to-end contact formation and intrachain diffusion coefficient. The data can be coherently interpreted on the basis of a simple chain model with backbone angles obtained from a library of coil segments of proteins and hard sphere repulsion at each C α position. In buffered water, we find that introducing charges in a glycine-rich sequence induces a mild chain swelling and a significant speed-up of the intrachain dynamics, whereas the removal of the glycines results in almost a two-fold increase of the chain volume and a drastic slowing down. In denaturants we observe a pronounced swelling of all the chains, with significant differences between the effect of urea and guanidinium chloride.

Effects of Denaturants on the Dynamics of Loop Formation in Polypeptides

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln) j-trp ( j = 1–9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4–0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from ∼0.6 nm to ∼0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50–400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.

Determination of Ultrafast Protein Folding Rates from Loop Formation Dynamics

Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.

Kinetics of Intramolecular Contact Formation in a Denatured Protein

Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of Csp Tm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein (∼10 4 s −1). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Förster resonance energy transfer (FRET) experiments.

Effects of Chain Stiffness on the Dynamics of Loop Formation in Polypeptides. Appendix: Testing a 1-Dimensional Diffusion Model for Peptide Dynamics

Quenching of the triplet state of tryptophan by cysteine provides an important new tool for measuring the rate at which a specific intramolecular contact is formed in disordered polypeptides. By measuring the viscosity dependence of the quenching rate, both the reaction-limited and the diffusion-limited quenching rates can be determined. The diffusion-limited rate corresponds to the rate of forming a short-range contact. The reaction-limited rate, which depends solely on the equilibrium end-to-end distribution, becomes essentially length-independent for short chains, providing clear-cut evidence that the chain is stiff. The length dependence of the reaction-limited rate can be accurately calculated using the distance dependence of the quenching rate determined at room temperature in a rigid glass, together with the end-to-end distance distribution for a wormlike chain having a persistence length of 0.6−0.7 nm. In addition, the length dependence of the diffusion-limited rate can also be reproduced by treat...

Peptide loop-closure kinetics from microsecond molecular dynamics simulations in explicit solvent.

End-to-end contact formation rates of several peptides were recently measured by tryptophan triplet quenching (Lapidus et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7220). Motivated by these experiments, we study loop-closure kinetics for two peptides of different lengths, Cys-(Ala-Gly-Gln)n-Trp (n = 1, 2), in multiple all-atom explicit-solvent molecular dynamics simulations with different initial conditions and force fields. In 150 simulations of approximately 20 ns each, we collect data covering 1.0 and 0.8 micros for the penta-peptide simulated with the AMBER and CHARMM force fields, respectively, and about 0.5 micros each with the two force fields for the octa-peptide. These extensive simulations allow us to analyze the dynamics of peptides in the unfolded state with atomic resolution, thus probing early events in protein folding, and to compare molecular dynamics simulations directly with experiment. The calculated lifetimes of the tryptophan triplet state are in the range of 50-100 ns, in agreement with experimental measurements. However, end-to-end contacts form more rapidly, with characteristic times less than 10 ns. The contact formation rates for the two force fields are similar despite differences in the respective ensembles of peptide conformations.

Spin−Lattice Relaxation of the Tryptophan Triplet State Varies with Its Protein Environment

Spin−lattice relaxation (SLR) has been studied at 1.2 K in the photoexcited triplet state of tryptophan (Trp) in a number of protein sites in zero applied magnetic field. We find that there is a distribution of SLR rate constants in every case that indicates the partitioning into two populations, ns0 and nf0, with differing behavior. ns0 undergoes slow SLR with rate constants in the range found in crystalline matrixes, while the SLR of nf0 is at least 1−2 orders of magnitude faster. The former population has considerable spin alignment during optical pumping, decays nonexponentially, and gives rise to optically detected magnetic resonance (ODMR) responses at 1.2 K. The latter has negligible spin alignment, decays largely as a single exponential, and is ODMR silent. The rapid SLR of nf0 is attributed to coupling with disorder (tunneling) modes that modulate the electron−electron dipolar coupling, and whose efficiency varies with protein site. The less efficient SLR of ns0 is the result of direct interactio...

Dynamics of intramolecular contact formation in polypeptides: distance dependence of quenching rates in a room-temperature glass.

Quenching of the triplet state of tryptophan by cysteine is an important new tool for measuring the rate of forming a specific contact between amino acids in a polypeptide chain. To determine the length scale associated with this contact, tryptophan was embedded in a room-temperature glass containing a high concentration of cysteine. The decay of the triplet population is extended in time, consistent with a rate coefficient that decreases exponentially with distance. Solving the diffusion equation with this distant-dependent rate reproduces the observed bimolecular rates in water and shows that quenching at low viscosities takes place less than or similar to A from van der Waals contact between the tryptophan and cysteine.

Measuring the rate of intramolecular contact formation in polypeptides.

Formation of a specific contact between two residues of a polypeptide chain is an important elementary process in protein folding. Here we describe a method for studying contact formation between tryptophan and cysteine based on measurements of the lifetime of the tryptophan triplet state. With tryptophan at one end of a flexible peptide and cysteine at the other, the triplet decay rate is identical to the rate of quenching by cysteine. We show that this rate is also close to the diffusion-limited rate of contact formation. The length dependence of this end-to-end contact rate was studied in a series of Cys-(Ala-Gly-Gln)(k)-Trp peptides, with k varying from 1 to 6. The rate decreases from approximately 1/(40 ns) for k = 1 to approximately 1/(140 ns) for k = 6, approaching the length dependence expected for a random coil (n(-3/2)) for the longest peptides.

Band-Shape Analysis of Delayed Slow-Passage ODMR in the Photoexcited Triplet State of Tryptophan

Delayed slow passage optically detected magnetic resonance (ODMR) band shapes of the triplet state of tryptophan, lysyl-tryptophanyl-lysine, N-acetyl tryptophanamide, and ribonuclease T1 from A. oryzae (RNaseT1) in low-temperature aqueous glass are analyzed using data obtained from independent microwave-induced delayed phosphorescence (MIDP) measurements as fixed input parameters. This method which uses only the band center frequency, Gaussian width, overall amplitude, and baseline position as variables is compared with a previously employed empirical analysis (Wu, J. Q.; Ozarowski, A.; Davis, S. K.; Maki, A. H. J. Phys. Chem. 1996, 100, 11496) that requires many more variables. Comparable quality of fit is found for the new, more rigorous method for each band except for the Tx ↔ Tz (D−E) ODMR transition which (with the exception of RNaseT1) deviates systematically when using the fixed kinetic parameters. The quality of fit of this band becomes comparable to that of the multiparameter empirical method upon introduction of dispersion in kx, which assumes values between −1 and −2 s-1/GHz. The sign and magnitude of this dispersion are consistent with previous kinetic data. Band centers and widths are altered only slightly from those obtained by the earlier empirical analysis.

Characterization of transient species in laser photolysis of aromatic amino acids using acetone as photosensitizer

The photochemical processes of aromatic amino acids were investigated in aqueous solution using acetone as photosensitizer by KrF (248 nm) laser flash photolysis. Laser-induced transient species were characterized according to kinetic analysis and quenching experiments. The intermediates recorded were assigned to the excited triplet state of tryptophan, the radicals of tryptophan and tyrosine. The excited triplet state of tryptophan produced via a triplet-triplet excitation transfer and the radicals arising from electron transfer reaction has been identified. Neither electron transfer nor energy transfer between triplet acetone and phenylalanine can occur in photolysis of phenylalanine aqueous solution which contains acetone. Furthermore, triplet acetone-induced radical transformation: Trp/N *-Tyr+Trp-Tyr/O* was observed directly in photolysis of dipeptide (Trp-Tyr) aqueous solution containing acetone, and the transformation resulting from intramolecular electron transfer was suggested.