Ashok Sekhar

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers.

The importance of dynamics to biomolecular function is becoming increasingly clear. A description of the structure–function relationship must, therefore, include the role of motion, requiring a shift in paradigm from focus on a single static 3D picture to one where a given biomolecule is considered in terms of an ensemble of interconverting conformers, each with potentially diverse activities. In this Perspective, we describe how recent developments in solution NMR spectroscopy facilitate atomic resolution studies of sparsely populated, transiently formed biomolecular conformations that exchange with the native state. Examples of how this methodology is applied to protein folding and misfolding, ligand binding, and molecular recognition are provided as a means of illustrating both the power of the new techniques and the significant roles that conformationally excited protein states play in biology.

Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD12SH). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD12SH has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization. DOI: http://dx.doi.org/10.7554/eLife.07296.001

Mapping the conformation of a client protein through the Hsp70 functional cycle

Significance Hsp70 chaperones are key components of the cellular proteostasis network. The ATP-dependent interaction of Hsp70 with its substrates prevents aggregation and promotes their correct folding and maturation. Here we investigate the impact of Hsp70 binding on the conformation of a client substrate using nuclear magnetic resonance spectroscopy. Our experiments provide a model for Hsp70 action in which the substrate can adopt substantial amounts of secondary structure even in the globally unfolded Hsp70-bound state. Moreover, the substrate conformation does not evolve as a function of the Hsp70 nucleotide state, demonstrating that the large structural changes in Hsp70 resulting from ATP binding and hydrolysis do not perform conformational work on the bound substrate or lead to changes in client protein conformation. The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

Hsp70 biases the folding pathways of client proteins

Significance Hsp70 (70-kDa heat shock protein) chaperones bind cognate substrates to prevent their aggregation and guide them toward their correctly folded, functional states. Here we use NMR spectroscopy to understand how this is achieved by studying a complex of Hsp70 with a folding competent substrate. Using an NMR experiment presented here, we show that long-range transient contacts are established in the unfolded, unbound state of the substrate. These contacts are greatly attenuated in the bound form of the substrate that also exists as an unfolded ensemble. Our results establish that Hsp70 binding can significantly bias the folding mechanism of client substrate molecules toward pathways where secondary structure is first generated, followed by the establishment of longer-range interactions in a distance-dependent fashion. The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a 1H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation

Significance The cell is divided into compartments where specific biochemical functions are performed. These compartments can be delineated by membranes or through phase separation of proteins or protein and nucleic acids to form membraneless organelles. The latter situation occurs with an intrinsically disordered region of Ddx4, a major constituent of germ granules. The nature of the interior of membraneless organelles is poorly understood. Here, we use NMR to show that the intrinsically disordered Ddx4 region remains disordered and highly dynamic in the phase-separated state, while diffusing as slowly as a particle the size of a bacterial cell. Ddx4 molecules form a network of interactions on phase separation, providing an alternative environment to that found in membrane-encapsulated organelles. Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid “membraneless organelles” that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, Ddx4cond, diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing Ddx4cond to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of Ddx4cond have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in Ddx4cond are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in Ddx4cond are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.

Probing the Free Energy Landscape of the Fast-Folding gpW Protein by Relaxation Dispersion NMR

The topographic features of the free energy landscapes that govern the thermodynamics and kinetics of conformational transitions in proteins, which in turn are integral for function, are not well understood. This reflects the experimental challenges associated with characterizing these multidimensional surfaces, even for small proteins. Here we focus on a 62-residue protein, gpW, that folds very rapidly into a native structure with an α/β topology in which α-helices are at the N- and C-terminal ends of the molecule with a central β-hairpin positioned orthogonally to the helices. Using relaxation dispersion NMR spectroscopy to probe the conformational fluctuations in gpW at 1 °C, we found that the native state interconverts with a transiently formed, sparsely populated second state with a lifetime of 250 μs, consistent with the global folding-unfolding rate under these conditions. In this low-populated state, the β-hairpin is unfolded whereas the α-helices remain predominantly formed. Our results argue for a hierarchical stability of secondary structural elements and demonstrate the existence of a complex free energy landscape even in this small, fast-folding single-domain protein.

Probing conformational dynamics in biomolecules via chemical exchange saturation transfer: a primer

Although Chemical Exchange Saturation Transfer (CEST) type NMR experiments have been used to study chemical exchange processes in molecules since the early 1960s, there has been renewed interest in the past several years in using this approach to study biomolecular conformational dynamics. The methodology is particularly powerful for the study of sparsely populated, transiently formed conformers that are recalcitrant to investigation using traditional biophysical tools, and it is complementary to relaxation dispersion and magnetization transfer experiments that have traditionally been used to study chemical exchange processes. Here we discuss the concepts behind the CEST experiment, focusing on practical aspects as well, we review some of the pulse sequences that have been developed to characterize protein and RNA conformational dynamics, and we discuss a number of examples where the CEST methodology has provided important insights into the role of dynamics in biomolecular function.

Understanding the mechanism of proteasome 20S core particle gating

Significance The proteasome plays a critical role in regulating cellular homeostasis by degrading target protein substrates. Gating residues that are part of the barrel-like proteasome structure provide a barrier to prevent inadvertent proteolysis. With NMR spectroscopy, the mechanism by which the gates interconvert between open and closed states is studied. We show that collisions with water molecules provide a driving force for gate interconversion and that the process takes place via many small steps that involve protein segments smaller than 4 Å. The effect of cellular lysate on proteasome gating is explored, establishing that gating equilibria and kinetics are not perturbed relative to buffer solutions under conditions of similar viscosity. The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood. Here, this is investigated by using magnetization transfer NMR spectroscopy focusing on the 20S proteasome core particle from Thermoplasma acidophilum. We show from viscosity-dependent proteasome gating kinetics that frictional forces originating from random solvent motions are critical for driving the gating process. Notably, a small effective hydrodynamic radius (EHR; <4Å) is obtained, providing a picture in which gate exchange proceeds through many steps involving only very small segment sizes. A small EHR further suggests that the kinetics of gate interconversion will not be affected appreciably by large viscogens, such as macromolecules found in the cell, so long as they are inert. Indeed, measurements in cell lysate reveal that the gate interconversion rate decreases only slightly, demonstrating that controlled studies in vitro provide an excellent starting point for understanding regulation of 20S core particle function in complex, biologically relevant environments.

Defining a length scale for millisecond-timescale protein conformational exchange

Although atomic resolution 3D structures of protein native states and some folding intermediates are available, the mechanism of interconversion between such states remains poorly understood. Here we study the four-helix bundle FF module, which folds via a transiently formed and sparsely populated compact on-pathway intermediate, I. Relaxation dispersion NMR spectroscopy has previously been used to elucidate the 3D structure of this intermediate and to establish that the conformational exchange between the I and the native, N, states of the FF domain is driven predominantly by water dynamics. In the present study we use NMR methods to define a length scale for the FF I–N transition, namely the effective hydrodynamic radius (EHR) that provides an average measure of the size of the structural units participating in the transition at any given time. Our experiments establish that the EHR is less than 4 Å, on the order of the size of one to two amino acid side chains, much smaller than the FF domain hydrodynamic radius (13 Å). The small magnitude of the EHR provides strong evidence that the I–N interconversion does not proceed via the synchronous motion of large clusters of amino acid residues, but rather by the exposure/burial of one or two side chains from solvent at any given time. Because the hydration of small hydrophobic solutes (< 4 Å) does not involve considerable dewetting or disruption of the water–hydrogen bonding network, the FF domain I–N transition does not require appreciable changes to the structure of the surrounding water.

Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state. DOI: http://dx.doi.org/10.7554/eLife.28030.001