Glassy Dynamics and Memory Effects in an Intrinsically Disordered Protein Construct.

Glassy, nonexponential relaxations in globular proteins are typically attributed to conformational behaviors that are missing from intrinsically disordered proteins. Yet, we show that single molecules of a disordered-protein construct display two signatures of glassy dynamics, logarithmic relaxations and a Kovacs memory effect, in response to changes in applied tension. We attribute this to the presence of multiple independent local structures in the chain, which we corroborate with a model that correctly predicts the force dependence of the relaxation. The mechanism established here likely applies to other disordered proteins.

Nanoparticle Mobility over a Surface as a Probe for Weak Transient Disordered Peptide-Peptide Interactions.

Weak interactions form the core basis of a vast number of biological processes, in particular, those involving intrinsically disordered proteins. Here, we establish a new technique capable of probing these weak interactions between synthetic unfolded polypeptides using a convenient yet efficient, quantitative method based on single particle tracking of peptide-coated gold nanoparticles over peptide-coated surfaces. We demonstrate that our technique is sensitive enough to observe the influence of a single amino acid mutation on the transient peptide-peptide interactions. Furthermore, the effects of buffer salinity, which are expected to alter weak electrostatic interactions, are also readily detected and examined in detail. The method presented here has the potential to evaluate, in a high-throughput manner, weak interactions for a wide range of disordered proteins, polypeptides, and other biomolecules.

Intramolecular Diffusion in α-Synuclein: It Depends on How You Measure It.

Intramolecular protein diffusion, the motion of one part of the polypeptide chain relative to another part, is a fundamental aspect of protein folding and may modulate amyloidogenesis of disease-associated intrinsically disordered proteins. Much work has determined such diffusion coefficients using a variety of probes, but there has been an apparent discrepancy between measurements using long-range probes, such as fluorescence resonance energy transfer, and short-range probes, such as Trp-Cys quenching. In this work, we make both such measurements on the same protein, α-synuclein, and confirm that such discrepancy exists. Molecular dynamics simulations suggest that such differences result from a diffusion coefficient that depends on the spatial distance between probes. Diffusional estimates in good quantitative agreement with experiment are obtained by accounting for the distinct distance ranges probed by fluorescence resonance energy transfer and Trp-Cys quenching.

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.

Sequential Closure of Loop Structures Forms the Folding Nucleus during the Refolding Transition of the Escherichia coli Adenylate Kinase Molecule.

The ensemble of conformers of globular protein molecules immediately following transfer from unfolding to folding conditions is assumed to be collapsed though still disordered, as the first steps of the folding pathway are initiated. In order to test the hypothesis that long loop closure transitions are part of the initiation of the folding pathway, our groups are studying the initiation of the folding transition of a model protein by time-resolved excitation energy transfer (trFRET) detected fast kinetics experiments. Site-specific double labeling is used to study the timing of conformational transitions of individual loop forming chain segments at the microsecond time regime. Previously, it was shown that at least three long loops in the Escherichia coli adenylate kinase (AK) molecule close within the first 5 ms of folding of AK, while the main global folding transition occurs in a time regime of seconds. In order to enhance the time resolution of the kinetics experiments to the microsecond time regime and determine the rate of closure of the two N terminal loops (loop I residues 1-26 and loop II residues 29-72), we applied a continuous flow based double kinetics experiment. These measurements enabled us to obtain a microsecond series of transient time dependent distributions of distances between the ends of the labeled loops. Analysis of the trFRET experiments show that the N terminal loop (loop I) is closed within less than 60 µs after the initiation of refolding. Loop II is also mostly closed within that time step but shows an additional small reduction of the mean end-to-end distance in a second phase at a rate of 0.005 µs(-1). This second phase can either reflect tightening of a loosely closed loop in the ensemble of conformers or may reflect two subpopulations in the ensemble, which differ in the rate of closure of loop II, but not in the rate of closure of loop I. This study shows the very fast closure of long loops in the otherwise disordered backbone and fine details of the very early hidden pretransition state steps that are essential for the fast and efficient folding of the protein molecule.

Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements.

Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET) measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET) measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM), and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro7 and the second polypeptide was a flexible molecule consisting of (Gly-Ser)7 repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.

Ensemble and single-molecule detected time-resolved FRET methods in studies of protein conformations and dynamics.

Most proteins are nanomachines that are selected to execute specific functions and therefore should have some degree of flexibility. The driving force that excites specific motions of domains and smaller chain elements is the thermal fluctuations of the solvent bath which are channeled to selected modes of motions by the structural constraints. Consequently characterization of the ensembles of conformers of proteins and their dynamics should be expressed in statistical terms, i.e., determination of probability distributions of the various conformers. This can be achieved by measurements of time-resolved dynamic non-radiative excitation energy transfer (trFRET) within ensembles of site specifically labeled protein molecules. Distributions of intramolecular segmental end-to-end distances and their fast fluctuations can be determined, and fast and slow conformational transitions within selected sections of the molecule can be monitored and analyzed. Both ensemble and single-molecule detection methods can be applied for data collection. In combination with synchronization methods, time-resolved FRET was also used for studies of fast conformational transitions, in particular the folding/unfolding transitions.

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways.

The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a "bottom-up" mechanism. Non-local interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an "up-down" mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the "loop hypothesis", is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the time-resolved FRET-based "double kinetics" method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.

An instrument for fast acquisition of fluorescence decay curves at picosecond resolution designed for "double kinetics" experiments: application to fluorescence resonance excitation energy transfer study of protein folding.

The information obtained by studying fluorescence decay of labeled biopolymers is a major resource for understanding the dynamics of their conformations and interactions. The lifetime of the excited states of probes attached to macromolecules is in the nanosecond time regime, and hence, a series of snapshot decay curves of such probes might - in principle - yield details of fast changes of ensembles of labeled molecules down to sub-microsecond time resolution. Hence, a major current challenge is the development of instruments for the low noise detection of fluorescence decay curves within the shortest possible time intervals. Here, we report the development of an instrument, picosecond double kinetics apparatus, that enables recording of multiple fluorescence decay curves with picosecond excitation pulses over wide spectral range during microsecond data collection for each curve. The design is based on recording and averaging multiphoton pulses of fluorescence decay using a fast 13 GHz oscilloscope during microsecond time intervals at selected time points over the course of a chemical reaction or conformational transition. We tested this instrument in a double kinetics experiment using reference probes (N-acetyl-tryptophanamide). Very low stochastic noise level was attained, and reliable multi-parameter analysis such as derivation of distance distributions from time resolved FRET (fluorescence resonance excitation energy transfer) measurements was achieved. The advantage of the pulse recording and averaging approach used here relative to double kinetics methods based on the established time correlated single photon counting method, is that in the pulse recording approach, averaging of substantially fewer kinetic experiments is sufficient for obtaining the data. This results in a major reduction in the consumption of labeled samples, which in many cases, enables the performance of important experiments that were not previously feasible.

Fast subdomain folding prior to the global refolding transition of E. coli adenylate kinase: a double kinetics study.

The rate of folding of globular proteins depends on specific local and nonlocal intramolecular interactions. What is the relative role of these two types of interaction at the initiation of refolding? We address this question by application of a "double kinetics" method based on fast initiation of refolding of site specifically labeled protein samples and detection of the transient distributions of selected intramolecular distances by means of fast measurements of time-resolved fluorescence resonance energy transfer. We determined the distribution of the distance between the ends of a 44-chain segment that includes the AMP(bind) domain, by labeling residues 28 and 71, in Escherichia coli adenylate kinase (AK) and the distribution of the distance between residues 18 and 203, which depends on the overall order of the molecule. That distribution shows two-state transition to the native intramolecular distance at the same rate as that of the cooperative refolding transition of the AK molecule. In sharp contrast, the distance distribution between residues 28 and 71 is already native like at the end of the dead-time of the mixing device. This fast formation of native short distance between two widely separated chain sections can be either dependent on fast folding of the AMP(bind) domain or a result of a very effective nonlocal interaction between specific short clusters of hydrophobic residues. Further experiments on studying the kinetics of folding of selected structural elements in the protein will help determination of the driving force of this early folding event.