Tomer Orevi

Early Closure of a Long Loop in the Refolding of Adenylate Kinase : A Possible Key Role of Non-Local Interactions in the Initial Folding Steps

Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions--that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This donor-acceptor pair of probes used with fluorescence resonance excitation energy transfer spectroscopy (FRET) is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude that a specific network of non-local interactions, the closure of one or several loops, can play an important role in determining the protein folding pathway at its early phases.

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.

Kinetics of Fast Changing Intramolecular Distance Distributions Obtained by Combined Analysis of FRET Efficiency Kinetics and Time-Resolved FRET Equilibrium Measurements

Detailed studies of the mechanisms of macromolecular conformational transitions such as protein folding are enhanced by analysis of changes of distributions for intramolecular distances during the transitions. Time-resolved Förster resonance energy transfer (FRET) measurements yield such data, but the more readily available kinetics of mean FRET efficiency changes cannot be analyzed in terms of changes in distances because of the sixth-power dependence on the mean distance. To enhance the information obtained from mean FRET efficiency kinetics, we combined the analyses of FRET efficiency kinetics and equilibrium trFRET experiments. The joint analysis enabled determination of transient distance distributions along the folding reaction both in cases where a two-state transition is valid and in some cases consisting of a three-state scenario. The procedure and its limits were tested by simulations. Experimental data obtained from stopped-flow measurements of the refolding of Escherichia coli adenylate kinase were analyzed. The distance distributions between three double-labeled mutants, in the collapsed transient state, were determined and compared to those obtained experimentally using the double-kinetics technique. The proposed method effectively provides information on distance distributions of kinetically accessed intermediates of fast conformational transitions induced by common relaxation methods.

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.

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.

Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

Abstract The protein folding problem would be considered “solved” when it will be possible to “read genes”, i.e., to predict the native fold of proteins, their dynamics, and the mechanism of fast folding based solely on sequence data. The long-term goal should be the creation of an algorithm that would simulate the stepwise mechanism of folding, which constrains the conformational space and in which random search for stable interactions is possible. Here, we focus attention on the initial phases of the folding transition starting with the compact disordered collapsed ensemble, in search of the initial sub-domain structural biases that direct the otherwise stochastic dynamics of the backbone. Our studies are designed to test the “loop hypothesis”, which suggests that fast closure of long loop structures by non-local interactions between clusters of mainly non-polar residues is an essential conformational step at the initiation of the folding transition of globular proteins. We developed and applied experimental methods based on time-resolved resonance excitation energy transfer (trFRET) measurements combined with fast mixing methods and studied the initial phases of the folding of Escherichia coli adenylate kinase (AK). A series of AK mutants were prepared, in which the ends of selected backbone segments that form long closed loops or secondary structure elements were labeled by donors and acceptors of excitation energy. The end-to-end distance distributions of such segments were determined under equilibrium and during the fast folding transitions. These experiments show that three out of seven long loops that were labeled in the AK molecule are closed very early in the transition. The N terminal 26-residue loop (loop I) is closed in <200 μs after the initiation of folding, while the β strand included in loop I is still disordered. The closure of the second 44-residue loop (loop II, which starts at the end of loop I) is also complete within <300 μs. Four other loops as well as five secondary structures of the CORE domain of AK (an α helix and four β strands) are formed at a late step, at a rate of 0.5±0.3 s–1, the rate of the cooperative folding of the molecule. These experiments reveal a hierarchically ordered pathway of folding of the AK molecule, ranging from microseconds to seconds. The results reviewed here, obtained mainly from studying a small number of model proteins, support the counterintuitive mechanism whereby non-local interactions are effective in the initiation of the folding pathways. The experiments presented demonstrate the importance of mapping the rates of sub-domain structural transitions along the folding transition, in situ, in the context of the other sections of the chain, whether folded or disordered. These experiments also show the power of the time-resolved FRET measurements in achieving this goal. A large body of data obtained by theoretical and experimental studies that support, or can accommodate, the loop hypothesis is reviewed. We suggest that mapping multiple sub-domain structural transitions during the refolding transition of many proteins using the approach presented here will refine the conclusions and help reveal some common principles of the initiation of the folding. To achieve this goal, the trFRET measurements should be combined with mutagenesis experiments where the role of selected residue clusters will be tested by perturbation mutations. Nevertheless, the solution of the protein folding problem depends on the application of many additional approaches, both experimental and theoretical, while the approach presented here is only a small section of the big puzzle.