Magic angle spinning NMR of proteins: high-frequency dynamic nuclear polarization and (1)H detection.

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.

Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus.

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure.

Intrinsic structural dynamics dictate enzymatic activity and inhibition.

Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors.

Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.

Solid-state NMR MAS CryoProbe enables structural studies of human blood protein vitronectin bound to hydroxyapatite.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.

Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Here, using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 µM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.

Measuring Protein-Ligand Binding by Hyperpolarized Ultrafast NMR.

Protein-ligand interactions can be detected by observing changes in the transverse relaxation rates of the ligand upon binding. The ultrafast NMR technique, which correlates the chemical shift with the transverse relaxation rate, allows for the simultaneous acquisition of R2 for carbon spins at different positions. In combination with dissolution dynamic nuclear polarization (D-DNP), where the signal intensity is enhanced by thousands of times, the R2 values of several carbon signals from unlabeled benzylamine are observable within a single scan. The hyperpolarized ultrafast chemical shift-R2 correlated experiment separates chemical shift encoding from the readout phase in the NMR pulse sequence, which allows it to beat the fundamental limit on the spectral resolution otherwise imposed by the sampling theorem. Applications enabled by the ability to measure multiple relaxation rates in a single scan include the study of structural properties of protein-ligand interactions.

Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies.

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Measuring Hydroxyl Exchange Rates in Glycans Using a Synergistic Combination of Saturation Transfer and Relaxation Dispersion NMR.

Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.

NMR spectroscopy, excited states and relevance to problems in cell biology - transient pre-nucleation tetramerization of huntingtin and insights into Huntington's disease.

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for analyzing three-dimensional structure and dynamics of macromolecules at atomic resolution. Recent advances have exploited the unique properties of NMR in exchanging systems to detect, characterize and visualize excited sparsely populated states of biological macromolecules and their complexes, which are only transient. These states are invisible to conventional biophysical techniques, and play a key role in many processes, including molecular recognition, protein folding, enzyme catalysis, assembly and fibril formation. All the NMR techniques make use of exchange between sparsely populated NMR-invisible and highly populated NMR-visible states to transfer a magnetization property from the invisible state to the visible one where it can be easily detected and quantified. There are three classes of NMR experiments that rely on differences in distance, chemical shift or transverse relaxation (molecular mass) between the NMR-visible and -invisible species. Here, I illustrate the application of these methods to unravel the complex mechanism of sub-millisecond pre-nucleation oligomerization of the N-terminal region of huntingtin, encoded by exon-1 of the huntingtin gene, where CAG expansion leads to Huntington's disease, a fatal autosomal-dominant neurodegenerative condition. I also discuss how inhibition of tetramerization blocks the much slower (by many orders of magnitude) process of fibril formation.

Prediction of order parameters based on protein NMR structure ensemble and machine learning.

The fast motions of proteins at the picosecond to nanosecond timescale, known as fast dynamics, are closely related to protein conformational entropy and rearrangement, which in turn affect catalysis, ligand binding and protein allosteric effects. The most used NMR approach to study fast protein dynamics is the model free method, which uses order parameter S2 to describe the amplitude of the internal motion of local group. However, to obtain order parameter through NMR experiments is quite complex and lengthy. In this paper, we present a machine learning approach for predicting backbone 1H-15N order parameters based on protein NMR structure ensemble. A random forest model is used to learn the relationship between order parameters and structural features. Our method achieves high accuracy in predicting backbone 1H-15N order parameters for a test dataset of 10 proteins, with a Pearson correlation coefficient of 0.817 and a root-mean-square error of 0.131.

Fluorine labelling for in situ 19F NMR in oriented systems.

The focus of this project is to take advantage of the large NMR chemical shift anisotropy of 19F to determine the orientation of fluorine labeled biomolecules in situ in oriented biological systems such as muscle. The difficulty with a single fluorine atom is that the orientation determined from a chemical shift is not singlevalued in the case of a fully anisotropic chemical shift tensor. The utility of a labeling approach with two fluorine labels in a fixed molecular framework where one of the labels has an axially symmetric chemical shift anisotropy such as a CF3 group and the other has a fully asymmetric chemical shift anisotropy such as 5-fluorotryptophan is evaluated. The result is that the orientation of the label can be determined straightforwardly from a single one-dimensional 19F NMR spectrum. The potential applications are widespread and not limited to biological applications.

Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( k e x , F I 1 ~ 550 s-1, k e x , F I 2 ~ 1200 s-1, k e x , I 1 I 2 ~ 5000 s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.

Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab.

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C', 13Cα, 13Cß resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs' higher order structures, dynamics and interactions, using solution-state NMR.

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

Short and long range 2D 15N-15N NMR correlations among peptide groups by novel solid state dipolar mixing schemes.

A recently developed homonuclear dipolar recoupling scheme, Adiabatic Linearly FREquency Swept reCOupling (AL FRESCO), was applied to record two-dimensional (2D) 15N-15N correlations on uniformly 15N-labeled GB1 powders. A major feature exploited in these 15N-15N correlations was AL FRESCO's remarkably low RF power demands, which enabled seconds-long mixing schemes when establishing direct correlations. These 15N-15N mixing schemes proved efficient regardless of the magic-angle spinning (MAS) rate and, being nearly free from dipolar truncation effects, they enabled the detection of long-range, weak dipolar couplings, even in the presence of strong short-range dipolar couplings. This led to a connectivity information that was significantly better than that obtained with spontaneously proton-driven, 15N spin-diffusion experiments. An indirect approach producing long-range 15N-15N correlations was also tested, relying on short (ms-long) 1HN-1HN mixings schemes while applying AL FRESCO chirped pulses along the 15N channel. These indirect mixing schemes produced numerous long-distance Ni-Ni±n (n = 2 - 5) correlations, that might be useful for characterizing three-dimensional arrangements in proteins. Once again, these AL FRESCO mediated experiments proved more informative than variants based on spin-diffusion-based 1HN-1HN counterparts.

A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy.

A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[15N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for 15N-labeling in HEK293 cells.

Uniform [13C,15N]-labeled and glycosylated IgG1 Fc expressed in Saccharomyces cerevisiae.

Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass E. coli culture. We developed a system utilizing Saccharomyces cerevisiae, [13C]-glucose, and [15N]-ammonium chloride with complexity comparable to E. coli. Here we report two vectors for expressing the crystallizable fragment (Fc) of IgG1 for secretion into the culture medium, utilizing the ADH2 or DDI2 promoters. We also report a strategy to optimize the expression yield using orthogonal Taguchi arrays. Lastly, we developed two different media formulations, a standard medium which provides 86-92% 15N and 30% 13C incorporation into the polypeptide, or a rich medium which provides 98% 15N and 95% 13C incorporation as determined by mass spectrometry. This advance represents an expression and optimization strategy accessible to experimenters with the capability to grow and produce proteins for NMR-based experiments using E. coli.