Schanda and Haran's 2024 Annual Review of Biophysics article on NMR and single-molecule FRET insights into fast protein motions poses a timescale dichotomy: simple physical considerations suggest that large-scale conformational transitions of proteins should be relatively fast, yet the functional cycles of many proteins (enzyme turnover, motor steps, signaling cycles) are often orders of magnitude slower, and current single-molecule and NMR methods are, in the authors' words, only 'starting to tackle this dichotomy' through a handful of case studies. The open core is a general mechanistic account of how the fast large-scale conformational transitions of proteins — motions in which loops, helices and domains change their structures or their relative positions — couple to the much slower functional cycles, answering the two questions the review itself asks: what the rate-determining factors for overall biological function are, and how important the fast motions are for the slower turnover.

Background

A folded protein is not a rigid object: it interconverts among many conformations, and these motions are central to its biological roles. Internal protein dynamics span an enormous range of timescales — local atomic fluctuations such as side-chain rotations and local fluctuations of loops occur on picosecond–nanosecond timescales; collective motions of helices and whole domains occur on microsecond–millisecond timescales; and the complete functional cycles of many proteins (enzyme turnover, motor steps, signaling cycles) take milliseconds to seconds. Two experimental techniques dominate the study of these motions. Nuclear magnetic resonance (NMR) spectroscopy measures fast picosecond–nanosecond fluctuations through the relaxation rates of nuclear spins, and slower microsecond–millisecond exchange between conformations through relaxation-dispersion experiments, which quantify how broadening of NMR lines varies with the spacing of refocusing radiofrequency pulses. Single-molecule Förster resonance energy transfer (smFRET) monitors the distance-dependent energy transfer between two fluorescent dyes attached to one protein molecule at a time, resolving conformational heterogeneity and the kinetics of individual transitions that ensemble methods average away. The review notes that no single method spans the full range of timescales, and it treats the complementary use of these techniques, together with in silico methods such as all-atom, accelerated or coarse-grained simulations and elastic network models, as valuable for integrating information obtained by different methods. The problem studied here is a puzzle stated explicitly by Schanda and Haran in a 2024 Annual Review of Biophysics: simple physical considerations suggest that large-scale conformational transitions should be relatively fast, yet the functional cycles of many proteins are often much slower — a dichotomy that, in their assessment, current methods are only beginning to tackle through case studies of enzymes and large protein machines. The 'fast' side of their dichotomy is specifically these large-scale transitions, in which secondary and tertiary structure elements (loops, helices and domains) change their structures or their positions with respect to one another: simple hydrodynamic considerations place the motion of a large structural element such as a domain on a nanosecond timescale, slowed to microseconds or longer by free-energy barriers on the potential energy surface, and the measurements the review surveys report microsecond-to-sub-millisecond motions, in some systems as slow as roughly 100 milliseconds. The review also stresses that the mismatch is not universal: while it emphasizes instances in which dynamics are faster than function, in several reported studies the rate of large-scale loop rearrangements was similar to the turnover rate. Several earlier results define the edges of this question, without answering it in general. First, in protein folding, photon-by-photon smFRET trajectories showed that the transition path — the brief interval during which a molecule actually changes structure — lasts less than 200 microseconds in a small two-state protein, more than 10,000 times shorter than the mean waiting time between folding and unfolding events (Chung, Louis and Eaton, 2009). This demonstrates that fast conformational change can coexist with slow dynamics because transitions are rare activated events on a rugged energy landscape (the set of conformational states of a protein together with their energies); whether an analogous rare-event account extends to functional cycles is not established. Second, in the enzyme adenylate kinase, which catalyzes the reversible reaction ATP + AMP ⇌ 2 ADP by closing lid domains over its substrates, NMR showed that picosecond–nanosecond fluctuations in the hinge regions facilitate the slower lid motions that produce the catalytically competent state, and the authors proposed that this hierarchy of timescales is a general characteristic of protein energy landscapes — a hypothesis that has been verified only in individual systems (Henzler-Wildman et al., 2007; Henzler-Wildman and Kern, 2007). The source review cites this hierarchy hypothesis as background rather than as its open target; adenylate kinase is, however, one of its case studies, where domain closure in the presence of substrates was found to be completed in just a few tens of microseconds, two orders of magnitude faster than turnover. Third, for allostery — communication between distant sites in a protein — ensemble models based on local unfolding (transient, partial loss of structure) explain many functional responses as redistribution of the populations of the conformational ensemble, a thermodynamic account that deliberately abstracts away kinetic timescales (Millard et al., 2025). The mechanistic vocabulary available for such questions — conformational selection (binding shifts the equilibrium toward a pre-existing minor conformation) versus induced fit (binding deforms the protein), dynamic allostery, kinetic gating — remains a collection of case-level explanations rather than a predictive theory. What is missing, and what the 2024 review identifies as the open frontier, is a general account linking the fast large-scale conformational motions that NMR and smFRET measure to the slow functional cycles that biochemistry measures, and explaining what sets the overall rate.

Problem Statement

Resolve the timescale dichotomy of protein dynamics as posed by Schanda and Haran (2024): provide a general, experimentally grounded mechanistic account of how the fast large-scale conformational transitions of proteins — motions in which secondary and tertiary structure elements (loops, helices and domains) change their structures or their positions with respect to one another, which simple physical considerations place on nanosecond-to-microsecond timescales and which NMR and single-molecule FRET measurements in the reviewed systems observe on microsecond-to-sub-millisecond timescales, in some cases up to roughly 100 milliseconds — are coupled to the much slower (millisecond-to-second) functional cycles of proteins, namely enzyme turnover, molecular-machine and motor steps, and signaling cycles. The account must address the two questions the review itself asks. First, what are the rate-determining factors for overall biological function: why are the functional cycles of many proteins often orders of magnitude slower than the rates at which the relevant large-scale conformational transitions can themselves occur, and under what conditions is turnover governed by the kinetics of the conformational transitions, by the equilibrium population of the catalytically competent state, or by other steps such as rare activated events on the energy landscape, rate-limiting chemistry, ligand binding or product release, and allosteric gating? The review's illustrative two-regime model writes the overall turnover rate constant as ktot=kI→Ak_{\mathrm{tot}} = k_{I \to A} when the transition from an inactive conformation II to the active conformation AA is rate-limiting, and as ktot=pAkchemk_{\mathrm{tot}} = p_A k_{\mathrm{chem}} when turnover is limited by the equilibrium population pAp_A of the active state together with the chemical rate kchemk_{\mathrm{chem}}. Second, how important are the fast motions for the slower turnover: what role do they play in the rate-limiting steps of function? The review emphasizes instances in which dynamics are faster than function while noting that this is not always the case — in several reported studies, for example on protein tyrosine phosphatases, the rate of large-scale loop rearrangements was similar to the turnover rate — and whether a large mismatch between the timescales of conformational dynamics and of functional cycles is a general design principle in proteins remains to be determined. The account must be consistent with the NMR and single-molecule FRET evidence the review draws on, for example domain closure in adenylate kinase completing in a few tens of microseconds, N-terminal-domain and pore-loop dynamics in ClpB, large-scale loop rearrangement in the TET2 aminopeptidase, DcpS, allosteric activation in HisHF, and p97/VCP. An answer resolves the task when a single account — rather than a separate explanation for each system — answers both questions above and is general across the systems the review surveys; the systems named here are illustrative examples of the evidence the account must be consistent with, not an exhaustive checklist.

References

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