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.

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells.

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble. Moreover, because the DNP polarization agent can be dispersed both homogenously and inhomogenously throughout the cellular biomass, in cell DNP-assisted solid state NMR experiments can report either quantitatively upon the structural ensemble or can preferentially report upon the structural ensemble with a spatial bias. Using DNP-assisted MAS NMR we establish that the spectra of purified α-syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. When the polarization agent is introduced into cells by electroporation and dispersed homogenously, a minority of the α-syn inside HEK293 cells adopts a highly α-helical rich conformation. Alteration of the spatial distribution of the polarization agent preferentially enhances the signal from molecules nearer to the cellular periphery, thus the α-helical rich population is preferentially adopted toward the cellular periphery. This demonstrates how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool for preferential reporting on specific structural ensembles, paving the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.

Conformational ensembles explain NMR spectra of frozen intrinsically disordered proteins.

Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble. We validated our simulations by comparing them to spectra of α-synuclein acquired under different experimental conditions. Our assignments of peaks to discrete dihedral angle populations suggest that structural constraints are attainable under cryogenic conditions. The ability to infer ensemble populations from peak shapes has important implications for DNP MAS NMR studies of proteins with regions of disorder in living cells because chemical shifts are the most accessible measured parameter.

Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells.

Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells. We find that nitroxide radicals are reduced with first order reduction kinetics by cell lysates at a rate of âˆ¼ 12% of the added nitroxide radical concentration per hour. We also found that electroporation delivered a consistent amount of AMUPol to intact cells and that nitroxide radicals are reduced just slightly more rapidly (∼15% per hour) by intact cells than by cell lysates. The two nitroxide radicals of AMUPol are reduced independently and this leads to considerable accumulation of the DNP-silent monoradical form of AMUPol, particularly in preparations of intact cells where nearly half of the AMUPol is already reduced to the DNP silent monoradical form at the earliest experimental time points. This confirms that the loss of the DNP-active biradical form of AMUPol is faster than the nitroxide reduction rate. Finally, we investigate the effect of adding N-ethyl maleimide, a well-known inhibitor of thiol (-SH) group-based reduction of nitroxide biradicals in cells, on AMUPol reduction, cellular viability, and DNP performance. Although pre-treatment of cells with NEM effectively inhibited the reduction of AMUPol, exposure to NEM compromised cellular viability and, surprisingly, did not improve DNP performance. Collectively, these results indicate that, currently, the most effective strategy to obtain high DNP enhancements for DNP-assisted in-cell NMR is to minimize room temperature contact times with cellular constituents and suggest that the development of bio-resistant polarization agents for DNP could considerably increase the sensitivity of DNP-assisted in-cell NMR experiments.

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs of in-cell experiments. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP-assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

In-Cell NMR of Intact Mammalian Cells Preserved with the Cryoprotectants DMSO and Glycerol Have Similar DNP Performance.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically-labeled proteins in complex environments and, with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. Prior work established that DNP MAS NMR is compatible with cellular viability. However, in that work, 15% glycerol, rather than the more commonly used 10% DMSO, was used as the cellular cryoprotectant. Moreover, incubation of cells cryoprotected 15% glycerol with the polarization agent, AMUPol, resulted in an inhomogeneous distribution of AMUPol through the cellular biomass, which resulted in a spatial bias of the NMR peak intensities. Because 10% DMSO is not only the most used cryoprotectant for mammalian cells, but also because DMSO is often used to improve delivery of molecules to cells, we sought to characterize the DNP performance of cells that were incubated with AMUPol and cryoprotected with 10% DMSO. We found that, like cells preserved with 15% glycerol, cells preserved with 10% DMSO retain high viability during DNP MAS NMR experiments if they are frozen at a controlled rate. However, DMSO did not improve the dispersion of AMUPol throughout the cellular biomass. Cells preserved with 15% glycerol and with 10% DMSO had similar DNP performance for both the maximal DNP enhancements as well as the inhomogeneous dispersion of AMUPol throughout the cellular biomass. Therefore, 10% DMSO and 15% glycerol are both appropriate cryoprotectant systems for DNP-assisted MAS NMR of intact viable mammalian cells.

Michler's hydrol blue elucidates structural differences in prion strains.

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

DNP-Assisted NMR Investigation of Proteins at Endogenous Levels in Cellular Milieu.

Structural investigations of biomolecules are typically confined to in vitro systems under extremely limited conditions. These investigations yield invaluable insights, but such experiments cannot capture important structural features imposed by cellular environments. Structural studies of proteins in their native contexts are not only possible using state-of-the-art sensitivity-enhanced (dynamic nuclear polarization, DNP) solid-state nuclear magnetic resonance (NMR) techniques, but these studies also demonstrate that the cellular context can and does have a dramatic influence on protein structure. In this chapter, we describe methods to prepare samples of isotopically labeled proteins at endogenous levels in cellular contexts alongside quality control methods to ensure that such samples accurately model important features of the cellular environment.

Amyloid fibrils embodying distinctive yeast prion phenotypes exhibit diverse morphologies.

Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Here, we investigate the structure of NM fibrils templated into the prion conformation with cellular lysates. Our electron microscopy studies reveal that NM fibrils that confer either a strong or a weak prion phenotype are both mixtures of thin and thick fibrils that result from differences in packing of the M domain. Strong NM fibrils have more thin fibrils and weak NM fibrils have more thick fibrils. Interestingly, both mass per length and solid state NMR reveal that the thin and thick fibrils have different underlying molecular structures in the prion strain variants that do not interconvert.

Aggregation and Fibril Structure of AßM01-42 and Aß1-42.

A mechanistic understanding of Aß aggregation and high-resolution structures of Aß fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aß peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aß1-42, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aß1-42 fusion protein in reasonable yield, with which we compared aggregation kinetics with AßM01-42 containing the additional methionine residue. The data revealed that Aß1-42 and AßM01-42 aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both AßM01-42 and Aß1-42 and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of AßM01-42 in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register.

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a ß-helical arrangement, whereas others suggest a parallel in-register organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13C- and 15N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems.

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.

Orientation of aromatic residues in amyloid cores: structural insights into prion fiber diversity.

Structural conversion of one given protein sequence into different amyloid states, resulting in distinct phenotypes, is one of the most intriguing phenomena of protein biology. Despite great efforts the structural origin of prion diversity remains elusive, mainly because amyloids are insoluble yet noncrystalline and therefore not easily amenable to traditional structural-biology methods. We investigate two different phenotypic prion strains, weak and strong, of yeast translation termination factor Sup35 with respect to angular orientation of tyrosines using polarized light spectroscopy. By applying a combination of alignment methods the degree of fiber orientation can be assessed, which allows a relatively accurate determination of the aromatic ring angles. Surprisingly, the strains show identical average orientations of the tyrosines, which are evenly spread through the amyloid core. Small variations between the two strains are related to the local environment of a fraction of tyrosines outside the core, potentially reflecting differences in fibril packing.

Distinct prion strains are defined by amyloid core structure and chaperone binding site dynamics.

Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic-angle spinning nuclear magnetic resonance spectroscopy, we provide a detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prion strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences in inheritance.

The role of conformational entropy in molecular recognition by calmodulin.

The physical basis for high-affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently used changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quantitative foundation. Here we calibrate an 'entropy meter' using an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin is surprisingly noncomplementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

Arylamide derivatives as peptidomimetic inhibitors of calmodulin.

[structure: see text] Many peptides bind to calmodulin (CaM) in a helical conformation. Here we describe a group of synthetic inhibitors of CaM based on an arylamide scaffold that is intended to mimic smMLCK, a CaM-binding helical peptide. Compound 1 showed a K(i) value of 7.10 +/- 1.48 nM in a fluorescence polarization assay that monitors the strong association of CaM and its peptide ligand mastoparan X. ((1)H,(15)N)-HSQC NMR spectroscopy experiments suggested that 1 binds to CaM in an analogous fashion to that of smMLCK.

Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB.

Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin. To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5'-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin. The enzyme from Escherichia coli is purified as a dimer that contains one [2Fe-2S]2+ cluster per monomer and can be reconstituted in vitro to contain an additional [4Fe-4S]2+ cluster per monomer. Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface. To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase. We find that both the [2Fe-2S]2+ cluster and the [4Fe-4S]2+ cluster must be present for tight substrate binding. Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet. The stoichiometry of binding is ca. 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer. AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at approximately 400 nm that we attribute to changes in the coordination environment of the [4Fe-4S]2+ cluster. Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex. This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB. Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5'-deoxyadenosyl radicals in the absence of the correct substrate.