NMR and Single-Molecule FRET Insights into Fast Protein Motions and Their Relation to Function.

Proteins often undergo large-scale conformational transitions, in which secondary and tertiary structure elements (loops, helices, and domains) change their structures or their positions with respect to each other. Simple considerations suggest that such dynamics should be relatively fast, but the functional cycles of many proteins are often relatively slow. Sophisticated experimental methods are starting to tackle this dichotomy and shed light on the contribution of large-scale conformational dynamics to protein function. In this review, we focus on the contribution of single-molecule Förster resonance energy transfer and nuclear magnetic resonance (NMR) spectroscopies to the study of conformational dynamics. We briefly describe the state of the art in each of each of these techniques and then point out their similarities and differences, as well as the relative strengths and weaknesses of each. Several case studies, in which the connection between fast conformational dynamics and slower function has been demonstrated, are then introduced and discussed. These examples include both enzymes and large protein machines, some of which have been studied by both NMR and fluorescence spectroscopies. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Single-molecule FRET probes allosteric effects on protein-translocating pore loops of a AAA+ machine.

AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using single-molecule FRET, we study the allosteric coupling between the pore loops and the two nucleotide-binding domains of ClpB (NBD1-2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore-loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines.

From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Förster Resonance Energy Transfer Study.

The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.

Perspective: How Fast Dynamics Affect Slow Function in Protein Machines.

Internal motions in proteins take place on a broad range of time- and space-scales. The potential roles of these dynamics in the biochemical functions of proteins have intrigued biophysicists for many years, and multiple mechanisms to couple motions to function have been proposed. Some of these mechanisms have relied on equilibrium concepts. For example, the modulation of dynamics was proposed to change the entropy of a protein, hence affecting processes such as binding. This so-called dynamic allostery scenario has been demonstrated in several recent experiments. Perhaps even more intriguing may be models that involve out-of-equilibrium operation, which by necessity require the input of energy. We discuss several recent experimental studies that expose such potential mechanisms for coupling dynamics and function. In Brownian ratchets, for example, directional motion is promoted by switching a protein between two free energy surfaces. An additional example involves the effect of microsecond domain-closure dynamics of an enzyme on its much slower chemical cycle. These observations lead us to propose a novel two-time-scale paradigm for the activity of protein machines: fast equilibrium fluctuations take place on the microsecond-millisecond time scale, while on a slower time scale, free energy is invested in order to push the system out of equilibrium and drive functional transitions. Motions on the two time scales affect each other and are essential for the overall function of these machines.

Microvillar Cartography: A Super-Resolution Single-Molecule Imaging Method to Map the Positions of Membrane Proteins with Respect to Cellular Surface Topography.

We describe microvillar cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, the need for carbon coating, and immunogold clustering, and is amenable to future developments involving, for example, live-cell imaging. This protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell-surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase.

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Dynamic correlations in lipid bilayer membranes over finite time intervals.

Recent single-molecule measurements [Schoch et al., Proc. Natl. Acad. Sci. U. S. A. 118, e2113202118 (2021)] have observed dynamic lipid-lipid correlations in membranes with submicrometer spatial resolution and submillisecond temporal resolution. While short from an instrumentation standpoint, these length and time scales remain long compared to microscopic molecular motions. Theoretical expressions are derived to infer experimentally measurable correlations from the two-body diffusion matrix appropriate for membrane-bound bodies coupled by hydrodynamic interactions. The temporal (and associated spatial) averaging resulting from finite acquisition times has the effect of washing out correlations as compared to naive predictions (i.e., the bare elements of the diffusion matrix), which would be expected to hold for instantaneous measurements. The theoretical predictions are shown to be in excellent agreement with Brownian dynamics simulations of experimental measurements. Numerical results suggest that the experimental measurement of membrane protein diffusion, in complement to lipid diffusion measurements, might help to resolve the experimental ambiguities encountered for certain black lipid membranes.

Fast dynamics shape the function of the AAA+ machine ClpB: lessons from single-molecule FRET spectroscopy.

It has been recently shown that in some proteins, tertiary-structure dynamics occur surprisingly fast, that is on the microsecond or sub-millisecond time scales. In this State of the Art Review, we discuss how such ultrafast domain motions relate to the function of caseinolytic peptidase B (ClpB), a AAA+ disaggregation machine. ClpB is a large hexameric protein that collaborates with cellular chaperone machinery to rescue protein chains from aggregates. We used single-molecule FRET spectroscopy to capture the dynamics of essential structural elements within this machine. It was found that the middle domain of ClpB, known to act as its activator, toggles between two states much faster than the overall activity cycle of the protein, suggesting a novel mode of continuous, tunable switching. Motions of the N-terminal domain were observed to restrict the conformational space of the M domain in the absence of a substrate protein, thereby preventing it from tilting and spuriously activating ClpB. Finally, microsecond dynamics of pore loops responsible for substrate pulling through ClpB's central channel, together with their response to specific perturbations, point to a Brownian-ratchet mechanism for protein translocation. Based on our findings, we propose a two-time-scale model for the activity of ClpB, in which fast conformational dynamics affect slower functional steps, determined by ATP hydrolysis time. Future work on this and other proteins is likely to shed further light on the role of ultrafast dynamics on protein function.

Role of Repeated Conformational Transitions in Substrate Binding of Adenylate Kinase.

The catalytic cycle of the enzyme adenylate kinase involves large conformational motions between open and closed states. A previous single-molecule experiment showed that substrate binding tends to accelerate both the opening and the closing rates and that a single turnover event often involves multiple rounds of conformational switching. In this work, we showed that the repeated conformational transitions of adenylate kinase are essential for the relaxation of incorrectly bound substrates into the catalytically competent conformation by combining all-atom and coarse-grained molecular simulations. In addition, free energy calculations based on all-atom and coarse-grained models demonstrated that the enzyme with incorrectly bound substrates has much a lower free energy barrier for domain opening compared to that with the correct substrate conformation, which may explain the the acceleration of the domain opening rate by substrate binding. The results of this work provide mechanistic understanding to previous experimental observations and shed light onto the interplay between conformational dynamics and enzyme catalysis.

The Effect of Time Resolution on Apparent Transition Path Times Observed in Single-Molecule Studies of Biomolecules.

Single-molecule experiments have now achieved a time resolution allowing observation of transition paths, the brief trajectory segments where the molecule undergoing an unfolding or folding transition enters the energetically or entropically unfavorable barrier region from the folded/unfolded side and exits to the unfolded/folded side, thereby completing the transition. This resolution, however, is yet insufficient to identify the precise entrance/exit events that mark the beginning and the end of a transition path: the nature of the diffusive dynamics is such that a molecular trajectory will recross the boundary between the barrier region and the folded/unfolded state, multiple times, at a time scale much shorter than that of the typical experimental resolution. Here we use theory and Brownian dynamics simulations to show that, as a result of such recrossings, the apparent transition path times are generally longer than the true ones. We quantify this effect using a simple model where the observed dynamics is a moving average of the true dynamics and discuss experimental implications of our results.

Control of protein activity by photoinduced spin polarized charge reorganization.

Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learned about electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.

Plasmonic Cavities and Individual Quantum Emitters in the Strong Coupling Limit.

ConspectusThe interaction of emitters with plasmonic cavities (PCs) has been studied extensively during the past decade. Much of the experimental work has focused on the weak coupling regime, manifested most importantly by the celebrated Purcell effect, which involves a modulation of the spontaneous emission rate of the emitter due to interaction with the local electromagnetic density of states. Recently, there has been a growing interest in studying hybrid emitter-PC systems in the strong-coupling (SC) regime, in which the excited state of an emitter hybridizes with that of the PC to generate new states termed polaritons. This phenomenon is termed vacuum Rabi splitting (VRS) and is manifested in the spectrum through splitting into two bands.In this Account, we discuss SC with PCs and focus particularly on work from our lab on the SC of quantum dots (QDs) and plasmonic silver bowtie cavities. As bowtie structures demonstrate strong electric field enhancement in their gaps, they facilitate approaching the SC regime and even reaching it with just one to a few emitters placed there. QDs are particularly advantageous for such studies, due to their significant brightness and long lifetime under illumination. VRS was observed in our lab by optical dark-field microspectroscopy even in the limit of individual QDs. We further used electron energy loss spectroscopy, a near-field spectroscopic technique, to facilitate measuring SC not only in bright modes but also in subradiant, dark plasmonic modes. Dark modes are expected to live longer than bright modes and therefore should be able to store electromagnetic energy for longer times.Photoluminescence (PL) is another useful observable for probing the SC regime at the single-emitter limit, as shown by several laboratories. We recently used Hanbury Brown and Twiss interferometry to demonstrate the quantum nature of PL from QDs within PCs, verifying that the measurements are indeed from one to three QDs. Further spectroscopic studies of QD-PC systems in fact manifested several surprising features, indicating discrepancies between scattering and PL spectra. These observations pointed to the contribution of multiple excited states. Indeed, using model simulations based on an extended Jaynes-Cummings Hamiltonian, it was found that the involvement of a dark state of the QDs can explain the experimental findings. Given that bright and dark states couple to the cavity with different degrees of coupling strength, the PC affects in a different manner each excitonic state. This yields complex relaxation pathways and interesting dynamics.Future work should allow us to increase the QD-PC coupling deeper into the SC regime. This will pave the way to exciting applications including the generation of single-photon sources and studies of cavity-induced coherent interactions between emitters.

Correlated diffusion in lipid bilayers.

Lipid membranes are complex quasi-two-dimensional fluids, whose importance in biology and unique physical/materials properties have made them a major target for biophysical research. Recent single-molecule tracking experiments in membranes have caused some controversy, calling the venerable Saffman-Delbrück model into question and suggesting that, perhaps, current understanding of membrane hydrodynamics is imperfect. However, single-molecule tracking is not well suited to resolving the details of hydrodynamic flows; observations involving correlations between multiple molecules are superior for this purpose. Here dual-color molecular tracking with submillisecond time resolution and submicron spatial resolution is employed to reveal correlations in the Brownian motion of pairs of fluorescently labeled lipids in membranes. These correlations extend hundreds of nanometers in freely floating bilayers (black lipid membranes) but are severely suppressed in supported lipid bilayers. The measurements are consistent with hydrodynamic predictions based on an extended Saffman-Delbrück theory that explicitly accounts for the two-leaflet bilayer structure of lipid membranes.

Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation.

AAA+ ring­shaped machines, such as the disaggregation machines ClpB and Hsp104, mediate ATP-driven substrate translocation through their central channel by a set of pore loops. Recent structural studies have suggested a universal hand-over-hand translocation mechanism with slow and rigid subunit motions. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB during substrate threading, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond time scale and change their conformation upon interaction with substrate proteins in an ATP-dependent manner. Conformational dynamics of two of the pore loops strongly correlate with disaggregation activity, suggesting that they are the main contributors to substrate pulling. This set of findings is rationalized in terms of an ultrafast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process in ClpB and other AAA+ machines.

CCR7 signalosomes are preassembled on tips of lymphocyte microvilli in proximity to LFA-1.

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain.

ClpB is a tightly regulated AAA+ disaggregation machine. Each ClpB molecule is composed of a flexibly attached N-terminal domain (NTD), an essential middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains. The NTD is not well-characterized structurally and is commonly considered to serve as a dispensable substrate-binding domain. Here, we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB's NTD and reveal its unexpected autoinhibitory function. We find that the NTD fluctuates on the microsecond time scale, and these dynamics result in steric hindrance that limits the conformational space of the MD to restrict its tilting. This leads to significantly inhibited ATPase and disaggregation activities of ClpB, an effect that is alleviated upon binding of a substrate protein or the cochaperone DnaK. This entropic inhibition mechanism, which is mediated by ultrafast motions of the NTD and is not dependent on any strong interactions, might be common in related ATP-dependent proteases and other multidomain proteins to ensure their fast and reversible activation.

Complex plasmon-exciton dynamics revealed through quantum dot light emission in a nanocavity.

Plasmonic cavities can confine electromagnetic radiation to deep sub-wavelength regimes. This facilitates strong coupling phenomena to be observed at the limit of individual quantum emitters. Here, we report an extensive set of measurements of plasmonic cavities hosting one to a few semiconductor quantum dots. Scattering spectra show Rabi splitting, demonstrating that these devices are close to the strong coupling regime. Using Hanbury Brown and Twiss interferometry, we observe non-classical emission, allowing us to directly determine the number of emitters in each device. Surprising features in photoluminescence spectra point to the contribution of multiple excited states. Using model simulations based on an extended Jaynes-Cummings Hamiltonian, we find that the involvement of a dark state of the quantum dots explains the experimental findings. The coupling of quantum emitters to plasmonic cavities thus exposes complex relaxation pathways and emerges as an unconventional means to control dynamics of quantum states.

Improving the quality factors of plasmonic silver cavities for strong coupling with quantum emitters.

Plasmonic cavities (PCs) made of metallic nanostructures can concentrate electromagnetic radiation into an ultrasmall volume, where it might strongly interact with quantum emitters. In recent years, there has been much interest in studying such a strong coupling in the limit of single emitters. However, the lossy nature of PCs, reflected in their broad spectra, limits their quality factors and hence their performance as cavities. Here, we study the effect of the adhesion layer used in the fabrication of metal nanostructures on the spectral linewidths of bowtie-structured PCs. Using dark-field microspectroscopy, as well as electron energy loss spectroscopy, it is found that a reduction in the thickness of the chromium adhesion layer we use from 3 nm to 0.1 nm decreases the linewidths of both bright and dark plasmonic modes. We further show that it is possible to fabricate bowtie PCs without any adhesion layer, in which case the linewidth may be narrowed by as much as a factor of 2. Linewidth reduction increases the quality factor of these PCs accordingly, and it is shown to facilitate reaching the strong-coupling regime with semiconductor quantum dots.