Intramolecular Contact Formation Research Articles

In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non-native interactions.

We examine the influence of cellular interactions in all-atom models of a section of the Homo sapiens cytoplasm on the early folding events of the three-helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30-microsecond simulations. We did however capture the formation of all three helices in a compact native-like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta-sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild-type sequence. This study demonstrates that even fast-folding proteins can get stuck in non-native states in the cell, making them useful models for protein-chaperone interactions and early stages of aggregate formation relevant to cellular disease.

Photo-induced electron transfer between tryptophan and cysteine: pH dependence.

Photo-induced electron transfer (PET) between tryptophan (W) and cysteine (C) is used to measure intra-molecular contact formation times in intrinsically disordered proteins (IDPs) and polymers. In this technique, a unique W and C pair are introduced into the protein sequence, at specific positions. Following nanosecond UV laser pulse excitation the W is excited to its triplet state, and the excited state triplet population is monitored in time via transient absorption. Quenching of the W triplet state occurs via electron transfer when C contacts W, due to stochastic collisions occurring in the disordered chain. The measured W triplet relaxation times report on intra-molecular contact formation times. By measuring such relaxation times under varying pH conditions, our group studies how electrostatic interactions affect IDP conformational ensembles and dynamics1-3 (see poster on NTAIL by L. Otteson et al.). The quantitative interpretation of the data requires knowledge of the bi-molecular quenching rate between W and C, freely diffusing in solution, at different pH values.We present an experimental study of the pH-dependence of W-C bimolecular quenching rates in solution, comparing near-physiological pH (7.6) to low pH (4.0), a range that is useful for our IDP experiments. We find that while the natural relaxation rate of the W triplet state (in the absence of quencher) is significantly larger at this pH, the bimolecular quenching rate (in the presence of C) does not vary much. We discuss the reasons for this effect and the implications to studying IDP dynamics.

Photo-induced electron transfer between tryptophan and cysteine: Temperature dependence.

Photo-induced electron transfer (PET) is used in experimental techniques to measure intra-molecular contact formation rates in intrinsically disordered proteins (IDPs) and polymers. A nanosecond UV excitation is used to excite W to its triplet state, which is quenched by cysteine (C) (or cystine (C_C)) via electron transfer upon contact, due to stochastic collisions. When a W and C (or C_C) are introduced in the IDP sequence at given positions, the rate of quenching via PET reports on the rate of contact formation between those two positions in the IDP. Our group has been using this technique to reveal important information about the conformational ensembles and dynamics of IDPs (see poster on NTAIL by L. Otteson et al.). While in the past we have been able to collect a large amount of temperature-dependent PET data in IDPs, their quantitative interpretation is complicated by the intrinsic temperature-dependence of the quenching process itself. Accurate measurements of the temperature dependence of the electron transfer rate (kET), between W and C in solution, are missing. Similar to our previous study, where we used C_C as a quencher (Kodis et al. 2021), here we present an experimental study of bimolecular quenching rates between W and C, measured in aqueous solution as a function of viscosity and temperature. We obtain reaction-limited (kET) and diffusion-limited (kD) contributions to the quenching rate at different temperatures. From these, we obtain the activation energy for the electron transfer rate (kET) between W and C and compare it to the value we previously found using C_C as a quencher. We discuss the implications of our findings for temperature dependent PET measurements in IDPs.

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.

N-Terminal Decarboxylation as a Probe for Intramolecular Contact Formation in γ-Glu-(Pro)n-Met Peptides.

The kinetics of intramolecular-contact formation between remote functional groups in peptides with restricted conformational flexibility were examined using designed peptides with variable-length proline bridges. As probes for this motion, free radicals were produced using the •OH-induced oxidation at the C-terminal methionine residue of γ-Glu-(Pro)n-Met peptides (n = 0–3). The progress of the radicals’ motion along the proline bridges was monitored as the radicals underwent reactions along the peptides’ backbones. Of particular interest was the reaction between the sulfur atom located in the side chain of the oxidized Met residue and the unprotonated amino group of the glutamic acid moiety. Interactions between them were probed by the radiation-chemical yields (expressed as G values) of the formation of C-centered, α-aminoalkyl radicals (αN) on the Glu residue. These radicals were monitored directly or via their reaction with p-nitroacetophenone (PNAP) to generate the optically detected PNAP•– radical anions. The yields of these αN radicals were found to be linearly dependent on the number of Pro residues. A constant decrease by 0.09 μM J–1 per spacing Pro residue of the radiation-chemical yields of G(αN) was observed. Previous reports support the conclusion that the αN radicals in these cases would have to result from (S∴N)+-bonded cyclic radical cations that arose as a result from direct contact between the ends of the peptides. Furthermore, by analogy with the rate constants for the formation of intramolecularly (S∴S)+-bonded radical cations in Met-(Pro)n-Met peptides (J. Phys. Chem. B2016, 120, 973227513096), the rate constants for the formation of intramolecularly (S∴N)+-bonded radical cations are activated to the same extent for all of the γ-Glu-(Pro)n-Met peptides. Thus, the continuous decrease of G(αN) with the number of Pro residues (from 0 to 3) suggests that the formation of a contact between the S-atom in the C-terminal Met residue and the N-atom of a deprotonated N-terminal amino group of Glu is controlled in peptides with 0 to 3 Pro residues by the relative diffusion of the S•+ and unoxidized N-atom. The overall rate constants of cyclization to form the (S∴N)-bonded radical cations were estimated to be 3.8 × 106, 1.8 × 106, and 8.1 × 105 s–1 for peptides with n = 0, 1, and 2 Pro residues, respectively. If activation is the same for all of the peptides, then these rate constants are a direct indication for the end-to-end dynamics along the chain.

(E)-2-{[(2-Amino-phen-yl)imino]-meth-yl}-5-(benz-yl-oxy)phenol and (Z)-3-benz-yl-oxy-6-{[(5-chloro-2-hy-droxy-phen-yl)amino]-methyl-idene}cyclo-hexa-2,4-dien-1-one.

The title Schiff base compounds, C20H18N2O2 (I) and C20H16ClNO3 (II), were synthesized from 4-benz-yloxy-2-hy-droxy-benzaldehyde by reaction with 1,2-di-amino-benzene for (I), and condensation with 2-amino-4-chloro-phenol for (II). Compound (I) adopts the enol-imine tautomeric form with an E configuration about the C=N imine bond. In contrast, the o-hy-droxy Schiff base (II), is in the keto-imine tautomeric form with a Z configuration about the CH-NH bond. Neither mol-ecule is planar. In (I), the central benzene ring makes dihedral angles of 46.80 (10) and 78.19 (10)° with the outer phenyl-amine and phenyl rings, respectively, while for (II), the corresponding angles are 5.11 (9) and 58.42 (11)°, respectively. The mol-ecular structures of both compounds are affected by the formation of intra-molecular contacts, an O-H⋯N hydrogen bond for (I) and an N-H⋯O hydrogen bond for (II); each contact generates an S(6) ring motif. In the crystal of (I), strong N-H⋯O hydrogen bonds form zigzag chains of mol-ecules along the b-axis direction. Mol-ecules are further linked by C-H⋯π inter-actions and offset π-π contacts and these combine to form a three-dimensional network. The density functional theory (DFT) optimized structure of compound (II), at the B3LYP/6-311+G(d) level, confirmed that the keto tautomeric form of the compound, as found in the structure determination, is the lowest energy form. The anti-oxidant capacities of both compounds were determined by the cupric reducing anti-oxidant capacity (CUPRAC) process.

Simultaneous Determination of Two Subdomain Folding Rates Using the “Transfer-Quench” Method

The investigation of the mechanism of protein folding is complicated by the context dependence of the rates of intramolecular contact formation. Methods based on site-specific labeling and ultrafast spectroscopic detection of fluorescence signals were developed for monitoring the rates of individual subdomain folding transitions in situ, in the context of the whole molecule. However, each site-specific labeling modification might affect rates of folding of near-neighbor structural elements, and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring subdomain structures using a single mutant to facilitate resolution of the order and interdependence of such steps. Here, we report the development of the “Transfer-Quench” method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on Förster resonance energy transfer combined with fluorescence quenching. We placed the donor and acceptor at the loop ends, and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple-labeled mutant is monitored by the acceptor emission. The formation of nonlocal contact (loop closure) increases the time-dependent acceptor emission, while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the common model protein, the B domain of staphylococcal protein A. Only natural amino acids were used as probes, and thus possible structural perturbations were minimized. Tyr and Trp residues served as donor and acceptor at the ends of a long loop between helices I and II, and a Cys residue as a quencher for the acceptor. We found that the closure of the loop (segment 14–33) occurs with the same rate constant as the nucleation of helix HII (segment 33–29), in line with the nucleation-condensation model.

Expansion and Internal Friction in Unfolded Protein Chain

Similarities in global properties of homopolymers and unfolded proteins provide approaches to mechanistic description of protein folding. Here, hydrodynamic properties and relaxation rates of the unfolded state of carbonmonoxide-liganded cytochrome c (cyt-CO) have been measured using nuclear magnetic resonance and laser photolysis methods. Hydrodynamic radius of the unfolded chain gradually increases as the solvent turns increasingly better, consistent with theory. Curiously, however, the rate of intrachain contact formation also increases with an increasing denaturant concentration, which, by Szabo, Schulten, and Schulten theory for the rate of intramolecular contact formation in a Gaussian polymer, indicates growing intramolecular diffusion. It is argued that diminishing nonbonded atom interactions with increasing denaturant reduces internal friction and, thus, increases the rate of polypeptide relaxation. Qualitative scaling of the extent of unfolding with nonbonded repulsions allows for description of internal friction by a phenomenological model. The degree of nonbonded atom interactions largely determines the extent of internal friction.

Intramolecular Distances and Dynamics from the Combined Photon Statistics of Single-Molecule FRET and Photoinduced Electron Transfer

Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher. We then performed combined FRET/PET experiments with FRET donor- and acceptor-labeled polyproline peptides. The placement of a tryptophan residue into the polyglycylserine tail incorporated in the peptides allowed us to measure both FRET efficiencies and the nanosecond dynamics of contact formation between one of the fluorescent dyes and the quencher. Variation of the linker length between the polyproline and the Alexa dyes and in the position of the tryptophan residue demonstrates the sensitivity of this approach. Modeling of the combined photon statistics underlying the combined FRET and PET process enables the accurate analysis of both the resulting transfer efficiency histograms and the nanosecond fluorescence correlation functions. This approach opens up new possibilities for investigating single biomolecules with high spatial and temporal resolution.

Curcumin Prevents Aggregation in α-Synuclein by Increasing Reconfiguration Rate

α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-β component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.

Effect of a Single-Point Mutation on the Conformation and Dynamics of Islet Amyloid Polypeptide from Nanosecond-Resolved Intramolecular Contact Formation

Islet amyloid polypeptide (IAPP) is an intrinsically disordered protein involved in regulating glucose metabolism and gastric emptying. It plays a crucial role in beta cell failure in diabetes type II, where it accumulates in the form of amyloid fibers. Human IAPP (hIAPP) is arguably the most amyloidogenic naturally occurring peptide known so far. Recent work by Raleigh and coworkers shows that a single-point mutation (I26P) converts it into an effective in vitro amyloid inhibitor1,2. The I26P mutation has been proposed to inhibit fibril formation by affecting the ability of the disordered C terminal tail to rearrange into the amyloid fibril structure, while maintaining intact the conformational properties of the N-terminal region, putatively responsible for the formation of aggregate intermediates1,2. Though such mechanism has been proposed, the conformational and dynamical properties of the I26P monomer have not been experimentally determined yet.We use tryptophan triplet quenching by cystine to measure the rate of contact formation between the two ends of the I26P peptide. This technique has previously revealed conformational differences between rIAPP and hIAPP3. It allows detecting both changes in the equilibrium end-to-end distance distribution and in the diffusional dynamics of the end-to-end distance caused by fast reconfigurations of backbone dihedral angles. We compare results for I26P, hIAPP, rIAPP, and model worm like chain peptides of same length. We discuss the effect of proline substitutions on the conformation and dynamics of these intrinsically disordered proteins and their possible effect on amyloid aggregation.Footnotes1 Abedini A. et al. JACS 129 20072 Meng F. et al. JACS 132 20103 Vaiana SM et al. BiophysJ 97 2009

A General Polymer Model of Unfolded Proteins under Folding Conditions

There is increasing evidence that a polypeptide chain in solvent conditions that favor folding may have transient structure and is significantly more compact than a fully denatured chain. However, there is no sequence-dependent model to capture such interactions. In this work, we present a simple and computationally inexpensive model based on a wormlike chain with excluded volume. The probability distribution of millions of such chains is reweighted to bias compact conformations in which residues of similar hydrophobicity are located near each other. This model, which has one adjustable parameter, is fit to measured values of intramolecular contact formation, which has been shown to be extremely sensitive to various models of intrachain distances. We show that under various denaturant concentrations, there is good convergence of the model for several different sequences with a wide range of dynamics. We also show that this model quantitatively predicts paramagnetic resonance enhancement (PRE) measurements with no adjustable parameters. Therefore a simple, probabilistic model that accounts for sequence-specific interactions may give a more realistic starting point for predictions of protein folding.

Connecting Unfolded Protein Dynamics and Aggregation

Solving the “protein folding problem” will require a better understanding of the allowed conformations and dynamics of the unfolded state. Fundamentally, unfolded protein dynamics should be determined by the hydrophobic pattern of each sequence. As proof of this hypothesis, we have constructed a random wormlike chain model that is re-weighted to favor random conformations that have residues of similar hydrophobicity in close proximity. This model gives remarkable quantitative agreement with measurements of intramolecular contact formation for various sequences. Furthermore we have found that the intramolecular diffusion coefficient of various sequences in folding conditions vary by ∼ 3 orders of magnitude. We find such dynamics qualitatively agree with those predicted by molecular dynamics in implicit solvent. The fastest sequences are intrinsically disordered proteins and peptides and the slowest sequences are well-behaved proteins with folding times of at least 1 ms. In between these two regimes are sequences prone to aggregation in which intramolecular diffusion is just fast enough to expose hydrophobes to solvent long enough to form bimolecular interactions.

Dynamics and Statistical Properties of Disordered Proteins

To understand the full picture of protein folding, structural and dynamic properties of the unfolded ensemble of a protein under folding conditions need to be elucidated. This could give clues to the early stages and events in the folding process. In this work, we examined the end-to-end intramolecular contact formation rates by the technique of tryptophan triplet quenching by cysteine, and numerically modeled the conformational distributions that agree with the experimental results using Szabo, Schulten and Schulten (SSS) theory, which treats intramolecular diffusion as diffusion on a one dimensional potential of mean forces.

Collapse Of Rat And Human Amylin From Nanosecond-resolved Intramolecular Contact Formation

Amylin is a 37 residue peptide with hormone properties related to nutrient intake regulating glucose levels. It is found in the form of amyloid deposits in the β-cells of type II diabetic patients. Similar to α-syn and Aβ, it is an intrinsically disordered protein. Little is known about amylin's conformational properties in solution and their relation to function and aggregation.We have used triplet quenching to monitor the dynamics of end-to-end contact formation between the N-terminal disulfide loop of human and rat amylin and a C-terminal tryptophan. The quenching rates for both species increase significantly in aqueous buffer relative to 6M guanidinium chloride (GdmCl), indicating a decrease in the average end-to-end distance. Comparisons with control peptides suggest that backbone-backbone interactions, involving the N-terminal disulfide loop are the principal driving force for collapse in these peptides, rather than sidechain-sidechain hydrophobic interactions. Molecular dynamics simulations on the control sequences indicate that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop, reducing the length of the free chain by ∼ 2-fold. This structural feature may contribute to the functional role of the disulfide loop in amylin and in the larger family of calcitonin gene-related peptides. We discuss the newly observed differences between monomeric human and rat IAPP in solution and their possible relation to aggregation.

Interdomain Communication in Hepatitis C Virus Polymerase Abolished by Small Molecule Inhibitors Bound to a Novel Allosteric Site

The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic intervention against HCV. We have determined the crystal structures of the HCV polymerase complexed with two indole-based allosteric inhibitors at 2.3- and 2.4-Angstroms resolution. The structures show that these inhibitors bind to a site on the surface of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets, whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme, the inhibitor binding site is occupied by a small alpha-helix at the tip of the N-terminal loop that connects the fingers and thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral agents against this clinically relevant human pathogen.

Impact of the Acidic C-Terminal Region Comprising Amino Acids 109−140 on α-Synuclein Aggregation in Vitro

The aggregation of alpha-synuclein, involved in the pathogenesis of several neurodegenerative disorders such as Parkinson's disease, is enhanced in vitro by biogenic polyamines binding to the highly charged C-terminal region aa109-140. In this study, we investigated the influence of this region on the aggregation kinetics, monitored by thioflavin T binding and static light scattering, and morphology, assessed by electron microscopy, fluorescence microscopy, and turbidity, by comparing the effect of various solution conditions on the wild-type protein, the disease related mutants A53T and A30P, and two truncated variants, syn(1-108) and syn(1-124), lacking the complete or the C-terminal half of the polyamine binding site. In the presence of the intact C-terminus, aggregation was strongly retarded in physiological buffer. This inhibition of aggregation was overridden by (i) addition of spermine or MgCl(2) or lowering of pH, leading to strong charge shielding in the C-terminus or (ii) by truncation of aa125-140 or aa109-140. Addition of MgCl(2) or spermine or acidification were not effective in promoting aggregation of syn(1-108). The impact of the disease-related mutations on the aggregation kinetics was dependent on the solution conditions, with the aggregation propensity order A53T approximately wt > A30P at low ionic strength, but A53T > wt approximately A30P at high ionic strength, with exceedingly potent promotion of aggregation by the A53T mutation in the presence of spermine. In contrast to full-length alpha-synuclein aggregates, those formed from syn(1-108) did not exhibit a pronounced polymorphism. The effects of the C-terminus on aggregation cannot be rationalized merely by a contribution to the protein net charge, but rather suggest a specific role of aa109-140 in the regulation of aggregation, presumably involving formation of intramolecular contacts.

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.

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.

A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.