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, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] 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 [Formula: see text] 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 [Formula: see text] 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.

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.energy landscape | transiently and sparsely populated biomolecular states | invisible states | structure-function paradigmThe field of structural biology emerged from seminal X-ray diffraction studies of biomolecules such as DNA (1), myoglobin (2), hemoglobin (3), and lysozyme (4) that were carried out over 50 years ago. Since these landmark investigations, our knowledge of the relation between structure and function has greatly expanded, driven by significant improvements in both experimental and computational approaches and, of course, by the exponential increase in the number of structures that are now available. Despite tremendous advances, structural biology remains largely focused on studies of the lowest-energy conformational states of biomolecules, and although there are notable exceptions (5), the end game often remains the determination of a single static 3D structure that is then used as a starting point for understanding molecular function. It is becoming increasingly clear, however, that this narrow approach is not sufficient and that a description of molecular structure must take into account conformational fluctuations that can occur over a broad range of both time and length scales.The conformational space that can be explored by a given biomolecule is usually explained by invoking the concept of an energy landscape (6), a multidimensional hypersurface that governs the thermodynamics and kinetics of conformational transitions (7,8). Because biomolecular stability is dictated by the sum of a large number of attractive and repulsive interactions of similar strength, the resultant free-energy landscape is rugged (9), with the global minimum in the surface thought to correspond to the native state of the biomolecule. In addition, there are often local minima that are separated from the global minimum by free-energy barriers that can be overcome by thermal fluctuations. These low-lying conformationally excited states have remained elusive to quantitative investigation because their sparse population and transient nature complicates their study by conventional structural biology techniques that are so powerfu...

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

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.

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 DnaKbound 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 | protein folding | NMR | molecular chaperones | CEST T he Hsp70 chaperone system forms a central hub in the cellular proteostasis network, performing a large number of functions, all of which are predicated upon its relatively simple ATPdependent interaction with a client protein (1, 2). Hsp70 is a 70-kDa protein containing both nucleotide (NBD) and substrate (SBD) binding domains connected by a conserved linker (2). The beststudied Hsp70 is Escherichia coli DnaK, which recognizes an approximate seven-residue protein segment rich in large aliphatic hydrophobic residues flanked by positively charged amino acids (3). Client substrates enter the Hsp70 functional cycle by binding the ATP form of the chaperone, which has lower substrate affinity but faster binding and release rates compared with the ADP state (2). The crystal structure of ATP-DnaK (4, 5) shows that the two domains of Hsp70 are docked on each other, with the helical lid flanking the DnaK binding cleft in an open state. Subsequent ATP hydrolysis, stimulated by both Hsp40 and the bound substrate, locks the substrate in the binding pocket (2) and disengages the two domains of DnaK from each other, leading to major structural differences between the ATP-(4, 5) and ADP-bound (6) forms of the chaperone. Finally, ADP release, facilitated by nucleotide exchange factors (7), and rebinding of ATP free the bound substrate and reset the chaperone cycle.Although the structural transitions of DnaK and the role of allostery throughout the chaperone reaction cycle are becoming increasingly well understood (7), far less information is available on the substrate conformation in the DnaK-bound state. Our current view is derived primarily from studies on the isolated SBD of DnaK in conjunctio...

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 1 H-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.Hsp70 | protein folding | excited states | molecular chaperones | PRE T he 70-kDa heat shock protein (Hsp70) chaperone system is an important component of the cellular proteostasis machinery, serving as a central hub to channel client proteins along folding, refolding, maturation, disaggregation, and proteolytic pathways in cooperation with other chaperone assemblies such as Hsp90, Hsp104, and GroEL/ES (1-3). Central to Hsp70 function is its ATP-dependent interaction with client proteins, facilitated by Hsp40 cochaperones and nucleotide exchange factors (NEFs) (4). Hsp70 is a weak ATPase that recognizes and binds substrates at sites containing large aliphatic hydrophobic residues, Ile, Leu, and Val, flanked by positively charged amino acids such as Arg and Lys (5). Initial binding of substrate to the ATP-form of Hsp70 can occur directly, or via Hsp40, with rapid on/off kinetics that give rise to a weak overall affinity for the interaction. Subsequent ATP hydrolysis, stimulated by interactions with Hsp40 and substrate, leads to a large conformational change in the chaperone, locking the substrate in the Hsp70 bound state. The resulting complex is of high affinity with slow substrate on/off rates (2).Escherichia coli DnaK is the best studied of Hsp70 chaperones. It is a 70-kDa protein comprised of an N-terminal ATPase and a C-terminal substrate binding domain that communicate allosterically to couple ATP hydrolysis with substrate binding (3). Highresolution structures of ADP-(6) and ATP-DnaK (7, 8) establish that these two domains dock on to one another in the ATP-DnaK state, but become detached from each other in the ADP-bound form. In contrast to the detailed structural studies characterizing DnaK, little atomic...

Biological molecules are often highly dynamic, and this flexibility can be critical for function. The large range of sampled timescales and the fact that many of the conformers that are continually explored are only transiently formed and sparsely populated challenge current biophysical approaches. Solution nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for characterizing biomolecular dynamics in detail, even in cases where excursions involve short-lived states. Here, we briefly review a number of NMR experiments for studies of biomolecular dynamics on the microsecond-to-second timescale and focus on applications to protein and nucleic acid systems that clearly illustrate the functional relevance of motion in both health and disease.

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