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The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively. It is challenging to distinguish between these mechanisms and other types of conformational exchange because chemical-shift-mediated line broadening provides only conclusive evidence for ring flips only if the system can be moved from the slow- to intermediate/fast-exchange regime. Moreover, whenever the chemical shift difference between the two symmetry-related sites is close to zero, it is not generally possible to determine the exchange time scale. Here we resolve these issues by measuring residual dipolar coupling (RDC)-mediated exchange contributions using NMR relaxation dispersion experiments on proteins dissolved in dilute liquid crystalline media. Excellent agreement is found between the experimental difference in RDC between the two symmetry-related sites and the value calculated from high-resolution X-ray structures, demonstrating that dynamics measured for F52 in the B1 domain of protein G reports on distinct, jumplike flips rather than other types of conformational exchange.

Aromatic residues form a significant part of the protein core, where they make tight interactions with multiple surrounding side chains. Despite the dense packing of internal side chains, the aromatic rings of phenylalanine and tyrosine residues undergo 180° rotations, or flips, which are mediated by transient and large-scale “breathing” motions that generate sufficient void volume around the aromatic ring. Forty years after the seminal work by Wagner and Wüthrich, NMR studies of aromatic ring flips are now undergoing a renaissance as a powerful means of probing fundamental dynamic properties of proteins. Recent developments of improved NMR methods and isotope labeling schemes have enabled a number of advances in addressing the mechanisms and energetics of aromatic ring flips. The nature of the transition states associated with ring flips can be described by thermodynamic activation parameters, including the activation enthalpy, activation entropy, activation volume, and also the isothermal volume compressibility of activation. Consequently, it is of great interest to study how ring flip rate constants and activation parameters might vary with protein structure and external conditions like temperature and pressure. The field is beginning to gather such data for aromatic residues in a variety of environments, ranging from surface exposed to buried. In the future, the combination of solution and solid-state NMR spectroscopy together with molecular dynamics simulations and other computational approaches is likely to provide detailed information about the coupled dynamics of aromatic rings and neighboring residues. In this Perspective, we highlight recent developments and provide an outlook toward the future.

Aromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic 1H R1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated 1H–13C spin pairs with vicinal 2H–12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the 3JHH couplings and limited chemical shift differences among 1H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, kflip, agree well with previous results from 13C R1ρ relaxation dispersion experiments, and yield 1H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic1H R1ρ relaxation dispersion experiment in combination with the site-selective 1H–13C/2H–12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s–1, and serve as a useful complement to previously developed 13C-based methods.

Proteins are dynamic entities that intermittently depart from their ground-state structures and undergo conformational transitions as a critical part of their functions. Central to understanding such transitions are the structural rearrangements along the connecting pathway, where the transition state plays a special role. Using NMR relaxation at variable temperature and pressure to measure aromatic ring flips inside a protein core, we obtain information on the structure and thermodynamics of the transition state. We show that the isothermal compressibility coefficient of the transition state is similar to that of short-chain hydrocarbon liquids, implying extensive local unfolding of the protein. Our results further indicate that the required local volume expansions of the protein can occur not only with a net positive activation volume of the protein, as expected from previous studies, but also with zero activation volume by compaction of remote void volume, when averaged over the ensemble of states.

Biochemical signaling is mediated by complexes between macromolecular receptors and their ligands, with the duration of the signal being directly related to the lifetime of the ligand–receptor complex. In the field of drug design, the recognition that drug efficacy in vivo depends on the lifetime of the drug–protein complex has spawned the concept of designing drugs with particular binding kinetics. To advance this field it is critical to investigate how the molecular details of designed ligands might affect the binding kinetics, as well as the equilibrium binding constant. Here we use protein NMR relaxation dispersion to determine linear free energy relationships involving the on- and off-rates and the affinity for a series of congeneric ligands targeting the carbohydrate recognition domain of galectin-3. Using this approach we determine the energy landscape and the position of the transition state along the reaction coordinate of protein–ligand binding. The results show that ligands exhibiting reduced off-rates achieve this by primarily stabilizing the bound state, but do not affect the transition state to any greater extent. The transition state forms early, that is, it is located significantly closer to the free state than to the bound state, suggesting a critical role of desolvation. Furthermore, the data suggest that different subclasses of ligands show different behavior with respect to these characteristics., NMR relaxation dispersion experiments reveal linear free energy relationships relating the binding constants to the lifetimes of protein–ligand complexes, showing that the transition state is located close to the free state.

Chemical shift tensors obtained from solid-state NMR spectroscopy are very sensitive reporters of structure and dynamics in proteins. While accurate (13)C and (15)N chemical shift tensors are accessible by magic angle spinning (MAS) NMR, their quantum mechanical calculations remain challenging, particularly for (15)N atoms. Here we compare experimentally determined backbone (13)C(α) and (15)N(H) chemical shift tensors by MAS NMR with hybrid quantum mechanics/molecular mechanics/molecular dynamics (MD-QM/MM) calculations for the carbohydrate-binding domain of galectin-3. Excellent agreement between experimental and computed (15)N(H) chemical shift anisotropy values was obtained using the Amber ff15ipq force field when solvent dynamics was taken into account in the calculation. Our results establish important benchmark conditions for improving the accuracy of chemical shift calculations in proteins and may aid in the validation of protein structure models derived by MAS NMR.

Plant hemoglobins, often referred to as phytoglobins, play important roles in abiotic stress tolerance. Several essential small physiological metabolites can be bound to these heme proteins. In addition, phytoglobins can catalyze a range of different oxidative reactions in vivo. These proteins are often oligomeric, but the degree and relevance of subunit interactions are largely unknown. In this study, we delineate which residues are involved in dimer formation of a sugar beet phytoglobin type 1.2 (BvPgb1.2) using NMR relaxation experiments. E. coli cells harboring a phytoglobin expression vector were cultivated in isotope-labeled (2H, 13C and 15N) M9 medium. The triple-labeled protein was purified to homogeneity using two chromatographic steps. Two forms of BvPgb1.2 were examined, the oxy-form and the more stable cyanide-form. Using three-dimensional triple-resonance NMR experiments, sequence-specific assignments for CN-bound BvPgb1.2 were achieved for 137 backbone amide cross-peaks in the 1H-15N TROSY spectrum, which amounts to 83% of the total number of 165 expected cross-peaks. A large proportion of the non-assigned residues are located in α-helixes G and H, which are proposed to be involved in protein dimerization. Such knowledge around dimer formation will be instrumental for developing a better understanding of phytoglobins’ roles in planta.

Amyloid fibril formation is a hallmark of neurodegenerative disease caused by protein aggregation. Oligomeric protein states that arise during the process of fibril formation often coexist with mature fibrils and are known to cause cell death in disease model systems. Progress in this field depends critically on development of analytical methods that can provide information about the mechanisms and species involved in oligomerization and fibril formation. Here, we demonstrate how the powerful combination of diffusion NMR and multilinear data analysis can efficiently disentangle the number of involved species, their kinetic rates of formation or disappearance, spectral contributions, and diffusion coefficients, even without prior knowledge of the time evolution of the process or chemical shift assignments of the various species. Using this method we identify oligomeric species that form transiently during aggregation of human superoxide dismutase 1 (SOD1), which is known to form misfolded aggregates in patients with amyotrophic lateral sclerosis. Specifically, over a time course of 42 days, during which SOD1 fibrils form, we detect the disappearance of the native monomeric species, formation of a partially unfolded intermediate in the dimer to tetramer size range, subsequent formation of a distinct similarly sized species that dominates the final spectrum detected by solution NMR, and concomitant appearance of small peptide fragments.

Post-translational methylation of lysine side chains is of great importance for protein regulation, including epigenetic control. Here, we present specific 13CHD2 labeling of dimethylated lysines a...

Multidimensional, heteronuclear NMR relaxation methods are used extensively to characterize the dynamics of biological macromolecules. Acquisition of relaxation datasets on proteins typically require significant measurement time, often several days. Accordion spectroscopy offers a powerful means to shorten relaxation rate measurements by encoding the 'relaxation dimension' into the indirect evolution period in multidimensional experiments. Time savings can also be achieved by nonuniform sampling (NUS) of multidimensional NMR data, which is used increasingly to improve spectral resolution or increase sensitivity per unit time. However, NUS is not commonly implemented in relaxation experiments, because most reconstruction algorithms are inherently nonlinear, leading to problems when estimating signal intensities, relaxation rate constants and their error bounds. We have previously shown how to avoid these shortcomings by combining accordion spectroscopy with NUS, followed by data reconstruction using sparse exponential mode analysis, thereby achieving a dramatic decrease in the total length of longitudinal relaxation experiments. Here, we present the corresponding transverse relaxation experiment, taking into account the special considerations required for its successfully implementation in the framework of the accordion-NUS approach. We attain the highest possible precision in the relaxation rate constants by optimizing the NUS scheme with respect to the Cramér-Rao lower bound of the variance of the estimated parameter, given the total number of sampling points and the spectrum-specific signal characteristics. The resulting accordion-NUS R1ρ relaxation experiment achieves comparable precision in the parameter estimates, compared to conventional CPMG R2 or spin-lock R1ρ experiments, while saving an order of magnitude in experiment time.

Allosteric communication pathways in the glucocorticoid receptor ligand-binding domain respond to ligand structure., Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

Bacteroides ovatus is a member of the human gut microbiota. The importance of this microbial consortium involves the degradation of complex dietary glycans mainly conferred by glycoside hydrolases. In this study we focus on one such catabolic glycoside hydrolase from B. ovatus. The enzyme, termed BoMan26A, is a β-mannanase that takes part in the hydrolytic degradation of galactomannans. The crystal structure of BoMan26A has previously been determined to reveal a TIM-barrel like fold, but the relation between the protein structure and the mode of substrate processing has not yet been studied. Here we report residue-specific assignments for 95% of the 344 backbone amides of BoMan26A. The assignments form the basis for future studies of the relationship between substrate interactions and protein dynamics. In particular, the potential role of loops adjacent to glycan binding sites is of interest for such studies.

Understanding the driving forces underlying molecular recognition is of fundamental importance in chemistry and biology. The challenge is to unravel the binding thermodynamics into separate contributions and to interpret these in molecular terms. Entropic contributions to the free energy of binding are particularly difficult to assess in this regard. Here we pinpoint the molecular determinants underlying differences in ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and molecular dynamics simulations followed by conformational entropy and grid inhomogeneous solvation theory (GIST) analyses. Using a pair of diastereomeric ligands that have essentially identical chemical potential in the unbound state, we reduced the problem of dissecting the thermodynamics to a comparison of the two protein-ligand complexes. While the free energies of binding are nearly equal for the R and S diastereomers, greater differences are observed for the enthalpy and entropy, which consequently exhibit compensatory behavior, ΔΔ H°(R - S) = -5 ± 1 kJ/mol and - TΔΔ S°(R - S) = 3 ± 1 kJ/mol. NMR relaxation experiments and molecular dynamics simulations indicate that the protein in complex with the S-stereoisomer has greater conformational entropy than in the R-complex. GIST calculations reveal additional, but smaller, contributions from solvation entropy, again in favor of the S-complex. Thus, conformational entropy apparently dominates over solvation entropy in dictating the difference in the overall entropy of binding. This case highlights an interplay between conformational entropy and solvation entropy, pointing to both opportunities and challenges in drug design.

Studies of protein−ligand binding often rely on dissolving the ligand in dimethyl sulfoxide (DMSO) to achieve sufficient solubility, and then titrating the ligand solution into the protein solution. As a result, the final protein−ligand solution contains small amounts of DMSO in the buffer. Here we report how the addition of DMSO impacts studies of protein conformational dynamics. We used 15N NMR relaxation to compare the rotational diffusion correlation time (τ C) of proteins in aqueous buffer with and without DMSO. We found that τ C scales with the viscosity of the water−DMSO mixture, which depends sensitively on the amount of DMSO and varies by a factor of 2 across the relevant concentration range. NMR relaxation studies of side chains dynamics are commonly interpreted using τ C as a fixed parameter, obtained from backbone 15N relaxation data acquired on a separate sample. Model‐free calculations show that errors in τ C, arising from mismatched DMSO concentration between samples, lead to significant errors in order parameters. Our results highlight the importance of determining τ C for each sample or carefully matching the DMSO concentrations between samples.

The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the 1H–15N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling. Electronic supplementary material The online version of this article (10.1007/s12104-018-9820-9) contains supplementary material, which is available to authorized users.

Journal of medicinal chemistry 61(3), 1164 - 1175 (2018). doi:10.1021/acs.jmedchem.7b01626, Symmetrical and asymmetrical fluorinated phenyltriazolyl-thiodigalactoside derivatives have been synthesized and evaluated as inhibitors of galectin-1 and galectin-3. Systematic tuning of the phenyltriazolyl-thiodigalactosides’ fluoro-interactions with galectin-3 led to the discovery of inhibitors with exceptional affinities (Kd down to 1–2 nM) in symmetrically substituted thiodigalactosides as well as unsurpassed combination of high affinity (Kd 7.5 nM) and selectivity (46-fold) over galectin-1 for asymmetrical thiodigalactosides by carrying one trifluorphenyltriazole and one coumaryl moiety. Studies of the inhibitor–galectin complexes with isothermal titration calorimetry and X-ray crystallography revealed the importance of fluoro-amide interaction for affinity and for selectivity. Finally, the high affinity of the discovered inhibitors required two competitive titration assay tools to be developed: a new high affinity fluorescent probe for competitive fluorescent polarization and a competitive ligand optimal for analyzing high affinity galectin-3 inhibitors with competitive isothermal titration calorimetry., Published by ACS, Washington, DC

The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.

We investigated galectin-3 binding to 3-benzamido-2-O-sulfo-galactoside and -thiodigalactoside ligands using a combination of site-specific mutagenesis, X-ray crystallography, computational approaches, and binding thermodynamics measurements. The results reveal a conformational variability in a surface-exposed arginine (R144) side chain in response to different aromatic C3-substituents of bound galactoside-based ligands. Fluorinated C3-benzamido substituents induced a shift in the side-chain conformation of R144 to allow for an entropically favored electrostatic interaction between its guanidine group and the 2-O-sulfate of the ligand. By contrast, binding of ligands with non-fluorinated substituents did not trigger a conformational change of R144. Hence, a sulfate-arginine electrostatic interaction can be tuned by the choice of ligand C3-benzamido structures to favor specific interaction modes and geometries. These results have important general implications for ligand design, as the proper choice of arginine-aromatic interacting partners opens up for ligand-controlled protein conformation that in turn may be systematically exploited in ligand design.

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.

Nonuniform sampling (NUS) of multidimensional NMR data offers significant time savings while improving spectral resolution or increasing sensitivity per unit time. However, NUS has not been widely used for quantitative analysis because of the nonlinearity of most methods used to model NUS data, which leads to problems in estimating signal intensities, relaxation rate constants, and their error bounds. Here, we present an approach that avoids these limitations by combining accordion spectroscopy and NUS in the indirect dimensions of multidimensional spectra and then applying sparse exponential mode analysis, which is well suited for analyzing accordion-type relaxation data in a NUS context. By evaluating the Cramer-Rao lower bound of the variances of the estimated relaxation rate constants, we achieve a robust benchmark for the underlying reconstruction model. Furthermore, we design NUS schemes optimized with respect to the information theoretical lower bound of the error in the parameters of interest, given a specified number of sampling points. The accordion-NUS method compares favorably with conventional relaxation experiments in that it produces identical results, within error, while shortening the length of the experiment by an order of magnitude. Thus, our approach enables rapid acquisition of NMR relaxation data for optimized use of spectrometer time or accurate measurements on samples of limited lifetime.

Using NMR and Monte Carlo (MC) methods, we investigate the stability and dynamics of superoxide dismutase 1 (SOD1) in homogeneous crowding environments, where either bovine pancreatic trypsin inhibitor (BPTI) or the B1 domain of streptococcal protein G (PGB1) serves as a crowding agent. By NMR, we show that both crowders, and especially BPTI, cause a drastic loss in the overall stability of SOD1 in its apo monomeric form. Additionally, we determine chemical shift perturbations indicating that SOD1 interacts with the crowder proteins in a residue-specific manner that further depends on the identity of the crowding protein. Furthermore, the specificity of SOD1-crowder interactions is reciprocal: chemical shift perturbations on BPTI and PGB1 identify regions that interact preferentially with SOD1. By MC simulations, we investigate the local unfolding of SOD1 in the absence and presence of the crowders. We find that the crowders primarily interact with the long flexible loops of the folded SOD1 monomer. The basic mechanisms by which the SOD1 β-barrel core unfolds remain unchanged when adding the crowders. In particular, both with and without the crowders, the second β-sheet of the barrel is more dynamic and unfolding-prone than the first. Notably, the MC simulations (exploring the early stages of SOD1 unfolding) and the NMR experiments (under equilibrium conditions) identify largely the same set of PGB1 and BPTI residues as prone to form SOD1 contacts. Thus, contacts stabilizing the unfolded state of SOD1 in many cases appear to form early in the unfolding reaction.

Magic angle spinning NMR spectroscopy is uniquely suited to probe the structure and dynamics of insoluble proteins and protein assemblies at atomic resolution, with NMR chemical shifts containing rich information about biomolecular structure. Access to this information, however, is problematic since accurate quantum mechanical calculation of chemical shifts in proteins remains challenging, particularly for (15)N(H). Here we report on isotropic chemical shift predictions for the carbohydrate recognition domain of microcrystalline galectin-3, obtained from using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, implemented using an automated fragmentation approach, and using very high resolution (0.86 Å lactose-bound and 1.25 Å apo form) X-ray crystal structures. The resolution of the X-ray crystal structure used as an input into the AF-NMR program did not affect the accuracy of the chemical shift calculations to any significant extent. Excellent agreement between experimental and computed shifts is obtained for (13)C(α) while larger scatter is observed for (15)N(H) chemical shifts, which are influenced to greater extent by electrostatic interactions, hydrogen bonding, and solvation.

FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on 15N and 2H NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔS = −18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔS = −26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetr...

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.

Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to match their targets better. We developed a computational approach for the structure-based design of repeat proteins that allows for adjustment of geometrical features like length, curvature, and helical twist. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeats (LRRs) from the ribonuclease inhibitor (RI) family that assemble into structures with a predefined geometry. The repeat proteins were built from self-compatible LRRs that are designed to interact to form highly curved and planar assemblies. We validated the geometrical design approach by engineering a ring structure constructed from 10 self-compatible repeats. Protein design can also be used to increase our structural understanding of repeat proteins. We use our design constructs to demonstrate that buried Cys play a central role for stability and folding cooperativity in RI-type LRR proteins. The computational procedure presented here may be used to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.

Protein dynamics on the microsecond-millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites have been used more sparingly. Aromatic side chains are useful probes of protein dynamics, because they are over-represented in protein binding interfaces, have important catalytic roles in enzymes, and form a sizable part of the protein interior. Here we present an off-resonance R 1ρ experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimization. Using selective excitation and inversion of the narrow component of the (13)C doublet, the experiment achieves significant sensitivity enhancement in terms of both signal intensity and the fractional contribution from exchange to transverse relaxation; additional signal enhancement is achieved by optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-selected R 1ρ experiment by measuring exchange parameters for Y23 in bovine pancreatic trypsin inhibitor at a temperature of 328 K, where the ring flip is in the fast exchange regime with a mean waiting time between flips of 320 μs. The determined chemical shift difference matches perfectly with that measured from the NMR spectrum at lower temperatures, where separate peaks are observed for the two sites. We further show that potentially complicating effects of strong scalar coupling between protons (Weininger et al. in J Phys Chem B 117: 9241-9247, 2013b) can be accounted for using a simple expression, and provide recommendations for data acquisition when the studied system exhibits this behavior. The present method extends the repertoire of relaxation methods tailored for aromatic side chains by enabling studies of faster processes and improved control over artifacts due to strong coupling.

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for (1)H spins in methyl (13)CHD2 groups, which improves the characterization of fast exchange processes. The influence of (1)H-(1)H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R 1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different (1)H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any (1)H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by (1)H and (13)C CPMG experiments. The present R 1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.

FKBP12, a small human enzyme, aids protein folding by catalyzing cis–trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s–1, similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNe2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is ...

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

Background Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide. Results We have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through β-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline β-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. Conclusions We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0300-3) contains supplementary material, which is available to authorized users.

We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.

Protein dynamics on the micro- to millisecond time scale is increasingly found to be critical for biological function, as demonstrated by numerous NMR relaxation dispersion studies. Methyl groups are excellent probes of protein interactions and dynamics because of their favorable NMR relaxation properties, which lead to sharp signals in the H-1 and C-13 NMR spectra. Out of the six different methyl-bearing amino acid residue types in proteins; methionine plays a special role because of its extensive side chain flexibility and the high polarizability of the sulfur atom. Methionine is over-represented:in many protein-protein recognition sites making the methyl group of this, residue type an important probe of the relationships among dynamics, Interactions, and biological function. Here we present a straightforward method to label methionine residues with specific (CHD2)-C-13 methyl isotopomers against a deuterated background. The resulting protein samples yield NMR spectra with improved sensitivity due to the essentially 100% population of the desired (CHD2)-C-13 methyl isotopomer, which is ideal for H-1 and C-13 spin relaxation experiments to investigate protein dynamics in general and conformational exchange in particular. We demonstrate the approach by measuring H-1 and C-13 CPMG relaxation dispersion for the nine methionines in calcium free calmodulin (apo-CaM). The results. show that the C- terminal domain, but not the N-terminal domain, of apo-CaM undergoes fast exchange between the ground state and a high-energy state. Since target proteins are known to bind specifically to the C-terminal domain of apo-CaM, we speculate that the high energy state might be involved in target binding through conformational selection. (Less)

Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively 13C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring 13C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached 1H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding–unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains. Electronic supplementary material The online version of this article (doi:10.1007/s10858-012-9656-z) contains supplementary material, which is available to authorized users.

Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of (13)C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring (13)C spins present in uniformly enriched proteins. Recently established protocols for specific (13)C labeling of aromatic side chains enable measurement of (13)C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R (1), R (2), and {(1)H}-(13)C NOE in specifically (13)C-labeled aromatic side chains. The optimized R (1) and R (2) experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond (13)C-(13)C scalar couplings or dipolar interactions with neighboring (1)H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.

Addition of a 50 mM mixture of L: -arginine and L: -glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded stale has a very low population. Here, we report pK(a) values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK(a) values were determined for carboxyl groups by monitoring their pH-dependent C-13 chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK(a) values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK(a) values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK(a) values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.

Here we present the 100% complete assignment chemical shift of non-labile H-1, N-15 and C-13 nuclei of Calbindin D-9k P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysN zeta as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge our assignments for Calbindin D-9k extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858-14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464-6465, 1994; Yamazaki et al. in Biochem 32:5656-5669, 1993b).

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein.

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the 15N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.

The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used (15)N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the (15)N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes.

A simple labeling approach is presented based on protein expression in [1-(13)C]- or [2-(13)C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C(alpha) sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta1) are produced with isolated (13)C spins (i.e., no (13)C-(13)C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C(beta) positions for 17 of the common 20 amino acids and there are no cases for which (13)C(alpha)-(13)CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone (15)N studies. The utility of the labeling is established by recording (13)C R (1rho) and CPMG-based experiments on a number of different protein systems.