Unfolded Ensemble Research Articles

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.

Unlocking the unfolded structure of ubiquitin: Combining time-resolved x-ray solution scattering and molecular dynamics to generate unfolded ensembles.

The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states.

Energy, water, and protein folding: A molecular dynamics-based quantitative inventory of molecular interactions and forces that make proteins stable.

Protein folding energetics can be determined experimentally on a case-by-case basis but it is not understood in sufficient detail to provide deep control in protein design. The fundamentals of protein stability have been outlined by calorimetry, protein engineering, and biophysical modeling, but these approaches still face great difficulty in elucidating the specific contributions of the intervening molecules and physical interactions. Recently, we have shown that the enthalpy and heat capacity changes associated to the protein folding reaction can be calculated within experimental error using molecular dynamics simulations of native protein structures and their corresponding unfolded ensembles. Analyzing in depth molecular dynamics simulations of four model proteins (CI2, barnase, SNase, and apoflavodoxin), we dissect here the energy contributions to ΔH (a key component of protein stability) made by the molecular players (polypeptide and solvent molecules) and physical interactions (electrostatic, van der Waals, and bonded) involved. Although the proteins analyzed differ in length, isoelectric point and fold class, their folding energetics is governed by the same quantitative pattern. Relative to the unfolded ensemble, the native conformations are enthalpically stabilized by comparable contributions from protein-protein and solvent-solvent interactions, and almost equally destabilized by interactions between protein and solvent molecules. The native protein surface seems to interact better with water than the unfolded one, but this is outweighed by the unfolded surface being larger. From the perspective of physical interactions, the native conformations are stabilized by van de Waals and Coulomb interactions and destabilized by conformational strain arising from bonded interactions. Also common to the four proteins, the sign of the heat capacity change is set by interactions between protein and solvent molecules or, from the alternative perspective, by Coulomb interactions.

Prevalence and mechanism of synergistic carboxylate-cation-water interactions in halophilic proteins

The cytoplasmic proteins of some halophilic organisms remain stable and functional at multimolar concentrations of KCl, i.e., under conditions that most mesophilic proteins cannot withstand. Their stability arises from their unusual amino acid composition. The most dramatic difference between halophilic and mesophilic proteins is that the former are rich in acidic amino acids. It has been proposed that one of the evolutionary driving forces for this difference is the occurrence of synergistic interactions between multiple acidic amino acids at the surface of the protein, the potassium cations in solution, and water. We investigate this possibility with molecular dynamics simulations, using high-quality force fields for the protein-water, protein-ion, and ion-ion interactions. We create a rigorous thermodynamic definition of interactions between acidic amino acids on proteins that can be used to distinguish between synergistic, noninteracting and interfering interactions. Our results demonstrate that synergistic interactions between neighboring acidic amino acids in halophilic proteins are frequent at multimolar KCl concentration. Synergistic interactions have an electrostatic origin, and are associated with stronger water-to-carboxylate hydrogen bonds than for acidic amino acids without synergistic interactions. Synergistic interactions are not observed in minimal systems of carboxylates, indicating that the protein environment is critical for their emergence. Our results demonstrate that synergistic interactions are neither associated with rigid amino acid orientations nor with highly structured and slow moving water networks, as had been originally proposed. Moreover, synergistic interactions can also be found in unfolded protein conformations. However, because these conformations are only a small subset of the unfolded state ensemble, synergistic interactions should contribute to the net stabilization of the folded state.

TMAO: Protecting proteins from feeling the heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 μs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules “herding” water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

Generation of unfolded outer membrane protein ensembles defined by hydrodynamic properties.

Outer membrane proteins (OMPs) must exist as an unfolded ensemble while interacting with a chaperone network in the periplasm of Gram-negative bacteria. Here, we developed a method to model unfolded OMP (uOMP) conformational ensembles using the experimental properties of two well-studied OMPs. The overall sizes and shapes of the unfolded ensembles in the absence of a denaturant were experimentally defined by measuring the sedimentation coefficient as a function of urea concentration. We used these data to model a full range of unfolded conformations by parameterizing a targeted coarse-grained simulation protocol. The ensemble members were further refined by short molecular dynamics simulations to reflect proper torsion angles. The final conformational ensembles have polymer properties different from unfolded soluble and intrinsically disordered proteins and reveal inherent differences in the unfolded states that necessitate further investigation. Building these uOMP ensembles advances the understanding of OMP biogenesis and provides essential information for interpreting structures of uOMP-chaperone complexes.

Structural basis of protein substrate processing by human mitochondrial high-temperature requirement A2 protease

The human high-temperature requirement A2 (HtrA2) protein is a trimeric protease that cleaves misfolded proteins to protect cells from stresses caused by toxic, proteinaceous aggregates, and the aberrant function of HtrA2 is closely related to the onset of neurodegenerative disorders. Our methyl-transverse relaxation optimized spectroscopy (TROSY)–based NMR studies using small-peptide ligands have previously revealed a stepwise activation mechanism involving multiple distinct conformational states. However, very little is known about how HtrA2 binds to protein substrates and if the distinct conformational states observed in previous peptide studies might be involved in the processing of protein clients. Herein, we use solution-based NMR spectroscopy to investigate the interaction between the N-terminal Src homology 3 domain from downstream of receptor kinase (drk) with an added C-terminal HtrA2-binding motif (drkN SH3-PDZbm) that exhibits marginal folding stability and serves as a mimic of a physiological protein substrate. We show that drkN SH3-PDZbm binds to HtrA2 via a two-pronged interaction, involving both its C-terminal PDZ-domain binding motif and a central hydrophobic region, with binding occurring preferentially via an unfolded ensemble of substrate molecules. Multivalent interactions between several clients and a single HtrA2 trimer significantly stimulate the catalytic activity of HtrA2, suggesting that binding avidity plays an important role in regulating substrate processing. Our results provide a thermodynamic, kinetic, and structural description of the interaction of HtrA2 with protein substrates and highlight the importance of a trimeric architecture for function as a stress-protective protease that mitigates aggregation.

Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein.

We investigate the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol (PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-molecule Förster resonance energy transfer measurements, we demonstrate that disordered CytR populates a well-folded minor conformation in its native ensemble, while the unfolded ensemble collapses and folds with an increase in crowder density independent of the crowder size. Employing a statistical-mechanical model, the effective reduction in the accessible conformational space of a residue in the unfolded state is estimated to be 10% at 300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG-temperature phase diagram thus constructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol-1, driving the equilibrium toward folded conformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.

Understanding the Folding Mediated Assembly of the Bacteriophage MS2 Coat Protein Dimers

The capsids of RNA viruses such as MS2 are great models for studying protein self-assembly because they are made almost entirely of multiple copies of a single coat protein (CP). Although CP is the minimal repeating unit of the capsid, previous studies have shown that CP exists as a homodimer (CP2) even in an acid-disassembled system, indicating that CP2 is an obligate dimer. Here, we investigate the molecular basis of this obligate dimerization using coarse-grained structure-based models and molecular dynamics simulations. We find that, unlike monomeric proteins of similar size, CP populates a single partially folded ensemble whose "foldedness" is sensitive to denaturing conditions. In contrast, CP2 folds similarly to single-domain proteins populating only the folded and the unfolded ensembles, separated by a prominent folding free energy barrier. Several intramonomer contacts form early, but the CP2 folding barrier is crossed only when the intermonomer contacts are made. A dissection of the structure of CP2 through mutant folding simulations shows that the folding barrier arises both from the topology of CP and the interface contacts of CP2. Together, our results show that CP2 is an obligate dimer because of kinetic stability, that is, dimerization induces a folding barrier and that makes it difficult for proteins in the dimer minimum to partially unfold and access the monomeric state without completely unfolding. We discuss the advantages of this obligate dimerization in the context of dimer design and virus stability.

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

The Cold-Unfolded State Is Expanded but Contains Long- and Medium-Range Contacts and Is Poorly Described by Homopolymer Models.

Cold unfolding of proteins is predicted by the Gibbs-Helmholtz equation and is thought to be driven by a strongly temperature-dependent interaction of protein nonpolar groups with water. Studies of the cold-unfolded state provide insight into protein energetics, partially structured states, and folding cooperativity and are of practical interest in biotechnology. However, structural characterization of the cold-unfolded state is much less extensive than studies of thermally or chemically denatured unfolded states, in large part because the midpoint of the cold unfolding transition is usually below freezing. We exploit a rationally designed point mutation (I98A) in the hydrophobic core of the C-terminal domain of the ribosomal protein L9 that allows the cold denatured state ensemble to be observed above 0 °C at near neutral pH and ambient pressure in the absence of added denaturants. A combined approach consisting of paramagnetic relaxation enhancement measurements, analysis of small-angle X-ray scattering data, all-atom simulations, and polymer theory provides a detailed description of the cold-unfolded state. Despite a globally expanded ensemble, as determined by small-angle X-ray scattering, sequence-specific medium- and long-range interactions in the cold-unfolded state give rise to deviations from homopolymer-like behavior. Our results reveal that the cold-denatured state is heterogeneous with local and long-range intramolecular interactions that may prime the folded state and also demonstrate that significant long-range interactions are compatible with expanded unfolded ensembles. The work also highlights the limitations of homopolymer-based descriptions of unfolded states of proteins.

Unfolding Dynamics of a Photoswitchable Helical Peptide

We present an atomistic force field for the azo-moiety of the photoswitchable FK-11-X peptide. We use the parameters to study the unfolding of the peptide through molecular dynamics simulations. The unfolded ensemble contains many different structures, ranging from a partially unfolded peptide to a fully unfolded structure. The averaged computed far-ultraviolet circular dichroism (CD) spectrum of the set of structures, which was simulated using the newly developed force field, agrees well with experiment. The rate of the simulated unfolding process was estimated to have a time constant of 5.80 ± 0.03 ns from the time evolution of the CD spectrum of the peptide, computed from the backbone conformations sampled over 40 simulated trajectories. Our estimated time constant is faster than, but not inconsistent with, previous experimental estimates from time-resolved infrared and optical rotatory dispersion spectroscopy.

Accurate Calculation of Barnase and SNase Folding Energetics Using Short Molecular Dynamics Simulations and an Atomistic Model of the Unfolded Ensemble: Evaluation of Force Fields and Water Models.

As proteins perform most cellular functions, quantitative understanding of protein energetics is required to gain control of biological phenomena. Accurate models of native proteins can be obtained experimentally, but the lack of equally fine models of unfolded ensembles impedes the calculation of protein folding energetics from first principles. Here, we show that an atomistic unfolded ensemble model, consisting of a few dozen conformations built from a protein sequence, can be used in conjunction with an X-ray structure of its native state to calculate accurately by difference the changes in enthalpy and heat capacity of the polypeptide upon folding. The calculation is done using molecular dynamics simulations, popular force fields, and water models, and for the two model proteins studied (barnase and SNase), the results agree within error or are very close to their experimentally determined properties. The enthalpy sampling of the unfolded ensemble is done through short 2 ns simulations that do not significantly modify the representative distribution of Rg of the starting conformations. The impressive accuracy obtained opens the possibility to investigate quantitatively systems or phenomena not amenable to experiment and paves the way for addressing the calculation of protein conformational stability (i.e., the change in Gibbs energy upon folding), a central goal of structural biology. So far, these calculated enthalpy and heat capacity changes, combined with the experimentally determined melting temperatures of the corresponding protein, allow us to reproduce the stability curves of both barnase and SNase.

Nonnative structure in a peptide model of the unfolded state of superoxide dismutase 1 (SOD1): Implications for ALS-linked aggregation

Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding free-energy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the local behavior of the corresponding sequences in the unfolded ensemble. Using time-resolved FRET, we observed that the peptide corresponding to the Loop VII-β8 sequence at the SOD1 C terminus was uniquely sensitive to denaturant. Utilizing a two-dimensional form of maximum entropy modeling, we demonstrate that the sensitivity to denaturant is the surprising result of a two-state-like transition from a compact to an expanded state. Variations of the peptide sequence revealed that the compact state involves a nonnative interaction between the disordered N terminus and the hydrophobic C terminus of the peptide. This nonnative intramolecular structure could serve as a precursor for intermolecular association and result in aggregation associated with ALS. We propose that this precursor would provide a common molecular target for therapeutic intervention in the dozens of ALS-linked SOD1 mutations.

High-Resolution Markov State Models for the Dynamics of Trp-Cage Miniprotein Constructed Over Slow Folding Modes Identified by State-Free Reversible VAMPnets.

State-free reversible VAMPnets (SRVs) are a neural network-based framework capable of learning the leading eigenfunctions of the transfer operator of a dynamical system from trajectory data. In molecular dynamics simulations, these data-driven collective variables capture the slowest modes of the dynamics and are useful for enhanced sampling and free energy estimation. In this work, we employ SRV coordinates as a feature set for Markov state model (MSM) construction. Compared to the current state-of-the-art, MSMs constructed from SRV coordinates are more robust to the choice of input features, exhibit faster implied time scale convergence, and permit the use of shorter lagtimes to construct higher kinetic resolution models. We apply this methodology to study the folding kinetics and conformational landscape of the Trp-cage miniprotein. Folding and unfolding mean first passage times are in good agreement with the prior literature, and a nine macrostate model is presented. The unfolded ensemble comprises a central kinetic hub with interconversions to several metastable unfolded conformations and which serves as the gateway to the folded ensemble. The folded ensemble comprises the native state, a partially unfolded intermediate "loop" state, and a previously unreported short-lived intermediate that we were able to resolve due to the high time resolution of the SRV-MSM. We propose SRVs as an excellent candidate for integration into modern MSM construction pipelines.

The Role of Protein Thermodynamics and Primary Structure in Fibrillogenesis of Variable Domains from Immunoglobulin Light Chains

Immunoglobulin light-chain amyloidosis is a protein aggregation disease that leads to proteinaceous deposits in a variety of organs in the body and, if untreated, ultimately results in death. The mechanisms by which light-chain aggregation occurs are not well understood. Here we have used solution NMR spectroscopy and biophysical studies to probe immunoglobulin variable domain λV6-57 VL aggregation, a process that appears to drive the degenerative phenotypes in amyloidosis patients. Our results establish that aggregation proceeds via the unfolded state. We identify, through NMR relaxation experiments recorded on the unfolded domain ensemble, a series of hotspots that could be involved in the initial phases of aggregate formation. Mutational analysis of these hotspots reveals that the region that includes K16-R24 is particularly aggregation prone. Notably, this region includes the site of the R24G substitution, a mutation that is found in variable domains of λ light-chain deposits in 25% of patients. The R24G λV6-57 VL domain aggregates more rapidly than would be expected on the basis of thermodynamic stability alone, while substitutions in many of the aggregation-prone regions significantly slow down fibril formation.

Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

Modeling pKas of unfolded proteins to probe structural models of unfolded state

Modeling unfolded states of proteins has implications for protein folding and stability. Since in unfolded state proteins adopt multiple conformations, any experimentally measured quantity is ensemble averaged, therefore the computed quantity should be ensemble averaged as well. Here, we investigate the possibility that one can model an unfolded state ensemble with the coil model approach, algorithm such as “flexible-meccano” [Ozenne V et al., Flexible-meccano: A tool for the generation of explicit ensemle descriptions of intrinsically disordered proteins and their associated experimental observables, Bioinformatics 28:1463–1470, 2012], developed to generate structures for intrinsically disordered proteins. We probe such a possibility by using generated structures to calculate pKas of titratable groups and compare with experimental data. It is demonstrated that even with a small number of representative structures of unfolded state, the average calculated pKas are in very good agreement with experimentally measured pKas. Also, predictions are made for titratable groups for which there is no experimental data available. This suggests that the coil model approach is suitable for generating 3D structures of unfolded state of proteins. To make the approach suitable for large-scale modeling, which requires limited number of structures, we ranked the structures according to their solvent accessible surface area (SASA). It is shown that in the majority of cases, the top structures with smallest SASA are enough to represent unfolded state.

Changes to the Intracellular Milieu Control the Population and Residual Structure of Unfolded Proteins

Proteins lacking a well-defined fold play important roles in cell signaling, proliferation, and disease. Due to their lack of tertiary structure, such proteins can be highly sensitive to the surrounding environment. The cellular environment is in constant flux: ionic strength, solute composition, crowding conditions, and other factors routinely change in response to both external and internal stimuli. Yet how these changes affect unfolded proteins in vivo is poorly understood. To understand the response of unfolded proteins to changes in the cellular milieu, we study the structure of the folded and unfolded state of a FRET-labeled glycolytic enzyme in as we perturb the intracellular environment. Live cell data is compared with in vitro measurements, and a computational model helps reveal the sensitivity of unfolded proteins to changes in crowding and solute concentrations in the cell. We show that changes to the intracellular environment can control not only the population of the unfolded state, but that the structure of the unfolded ensemble can itself be altered in the cell. We propose that unfolded proteins, and especially certain intrinsically disordered proteins, are designed to sense and respond to intracellular environment changes by altering their residual structure.

Measuring Diffusion Constants of Invisible Protein Conformers by Triple-Quantum 1 H CPMG Relaxation Dispersion.

Proteins are not locked in a single structure but often interconvert with other conformers that are critical for function. When such conformers are sparsely populated and transiently formed they become invisible to routine biophysical methods, however they can be studied in detail by NMR spin-relaxation experiments. Few experiments are available in the NMR toolkit, however, for characterizing the hydrodynamic properties of invisible states. Herein we describe a CPMG-based experiment for measuring translational diffusion constants of invisible states using a pulsed-field gradient approach that exploits methyl 1 H triple-quantum coherences. An example, involving diffusion of a sparsely populated and hence invisible unfolded protein ensemble is presented, without the need for the addition of denaturants that tend to destroy weak interactions that can be involved in stabilizing residual structure in the unfolded state.