Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C'-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.

Electron and Spin Delocalization in [Co6 Se8 (PEt3 )6 ]0/+1 Superatoms.

Molecular clusters can function as nanoscale atoms/superatoms, assembling into superatomic solids, a new class of solid-state materials with designable properties through modifications on superatoms. To explore possibilities on diversifying building blocks, here we thoroughly studied one representative superatom, Co6 Se8 (PEt3 )6 . We probed its structural, electronic, and magnetic properties and revealed its detailed electronic structure as valence electrons delocalize over inorganic [Co6 Se8 ] core while ligands function as an insulated shell. 59 Co SSNMR measurements on the core and 31 P, 13 C on the ligands show that the neutral Co6 Se8 (PEt3 )6 is diamagnetic and symmetric, with all ligands magnetically equivalent. Quantum computations cross-validate NMR results and reveal degenerate delocalized HOMO orbitals, indicating aromaticity. Ligand substitution keeps the inorganic core nearly intact. After losing one electron, the unpaired electron in [Co6 Se8 (PEt3 )6 ]+1 is delocalized, causing paramagnetism and a delocalized electron spin. Notably, this feature of electron/spin delocalization over a large cluster is attractive for special single-electron devices.

Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1.

T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn 2+ binds primarily to RRM3. Binding of Zn 2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long "daisy chained" structures, the addition of Zn 2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn 2+ . By occupying various "half" binding sites on RRM3 Zn 2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution.

Contribution of protein conformational heterogeneity to NMR lineshapes at cryogenic temperatures.

While low temperature NMR holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in E. coli DHFR at 105K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of N i+1 to Ψ i . With selective 15 N and 13 C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13 C'- 15 N correlation spectrum. For this unique amide we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin 114 ± 7 for the major peak, and 150 ± 8 and 164 ± 16° for the minor peak as contrasted with 118 for the X-ray crystal structure (and 118-130 for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low temperature NMR spectra. Significance Statement: Understanding protein conformational flexibility is essential for insights into the molecular basis of protein function and the thermodynamics of proteins. Here we investigate the ensemble of protein backbone conformations in a frozen protein freezing, which is likely a close representation for the ensemble in rapid equilibrium at room temperature. Various conformers are spectrally resolved due to the exquisite sensitivity of NMR shifts to local conformations, and NMR methods allow us to directly probe the torsion angles corresponding to each band of chemical shifts.

GTP-Bound Escherichia coli FtsZ Filaments Are Composed of Tense Monomers: a Dynamic Nuclear Polarization-Nuclear Magnetic Resonance Study Using Interface Detection.

FtsZ filaments are the major structural component of the bacterial Z ring and are drivers of bacterial division. Crystal structures for FtsZ from some Gram-positive bacteria in the presence of GTP analogs suggest the possibility of a high-energy, "tense" conformation. It remains important to elucidate whether this tense form is the dominant form in filaments. Using dynamic nuclear polarization (DNP) solid-state nuclear magnetic resonance (NMR) and differential isotopic labeling, we directly detected residues located at the intermonomer interface of GTP-bound wild-type (WT) Escherichia coli FtsZ filaments. We combined chemical shift prediction, homology modeling, and heteronuclear dipolar recoupling techniques to characterize the E. coli FtsZ filament interface and demonstrated that the monomers in active filaments assume a tense conformation. IMPORTANCE Bacterial replication is dependent on the cytoskeletal protein FtsZ, which forms filaments that scaffold and recruit other essential division proteins. While the FtsZ monomer is well studied across organisms, many questions remain about how the filaments form and function. Recently, a second monomer form was identified in Staphylococcus aureus that has far-reaching implications for FtsZ structure and function. However, to date, this form has not been directly observed outside S. aureus. In this study, we used solid-state NMR and dynamic nuclear polarization (DNP) to directly study the filaments of E. coli FtsZ to demonstrate that E. coli FtsZ filaments are primarily composed of this second, "tense" form of the monomer. This work is the first time GTP-bound, wild-type FtsZ filaments have been studied directly at atomic resolution and is an important step forward for the study of FtsZ filaments.

Rotating Frame Relaxation in Magic Angle Spinning Solid State NMR, a Promising Tool for Characterizing Biopolymer Motion.

Magic angle spinning NMR rotating frame relaxation measurements provide a unique experimental window into biomolecules dynamics, as is illustrated by numerous recent applications. We discuss experimental strategies for this class of experiments, with a particular focus on systems where motion-driven modulation of the chemical shift interaction is the main mechanism for relaxation. We also explore and describe common strategies for interpreting the data sets to extract motion time scale, activation energy, and angle or order parameters from rotating frame relaxation data. Using model free analysis and numerical simulations, including time domain treatment, we explore conditions under which it is possible to obtain accurate and precise information about the time scales of motions. Overall, with rapid technical advances in solid state NMR, there is a bright future for this class of studies.

High-Resolution Magic Angle Spinning NMR of KcsA in Liposomes: The Highly Mobile C-Terminus.

The structure of the transmembrane domain of the pH-activated bacterial potassium channel KcsA has been extensively characterized, yet little information is available on the structure of its cytosolic, functionally critical N- and C-termini. This study presents high-resolution magic angle spinning (HR-MAS) and fractional deuteration as tools to study these poorly resolved regions for proteoliposome-embedded KcsA. Using 1H-detected HR-MAS NMR, we show that the C-terminus transitions from a rigid structure to a more dynamic structure as the solution is rendered acidic. We make previously unreported assignments of residues in the C-terminus of lipid-embedded channels. These data agree with functional models of the C-terminus-stabilizing KcsA tetramers at a neutral pH with decreased stabilization effects at acidic pH. We present evidence that a C-terminal truncation mutation has a destabilizing effect on the KcsA selectivity filter. Finally, we show evidence of hydrolysis of lipids in proteoliposome samples during typical experimental timeframes.

Iterative Synthesis of Contorted Macromolecular Ladders for Fast-Charging and Long-Life Lithium Batteries.

We report here an iterative synthesis of long helical perylene diimide (hPDI[n]) nanoribbons with a length up to 16 fused benzene rings. These contorted, ladder-type conjugated, and atomically precise nanoribbons show great potential as organic fast-charging and long-lifetime battery cathodes. By tuning the length of the hPDI[n] oligomers, we can simultaneously modulate the electrical conductivity and ionic diffusivity of the material. The length of the ladders adjusts both the conjugation for electron transport and the contortion for lithium-ion transport. The longest oligomer, hPDI[6], when fabricated as the cathode in lithium batteries, features both high electrical conductivity and high ionic diffusivity. This electrode material exhibits a high power density and can be charged in less than 1 min to 66% of its maximum capacity. Remarkably, this material also has exceptional cycling stability and can operate for up to 10,000 charging-discharging cycles without any appreciable capacity decay. The design principles described here chart a clear path for organic battery electrodes that are sustainable, fast-charging, and long lasting.

Informing NMR experiments with molecular dynamics simulations to characterize the dominant activated state of the KcsA ion channel.

As the first potassium channel with an x-ray structure determined, and given its homology to eukaryotic channels, the pH-gated prokaryotic channel KcsA has been extensively studied. Nevertheless, questions related, in particular, to the allosteric coupling between its gates remain open. The many currently available x-ray crystallography structures appear to correspond to various stages of activation and inactivation, offering insights into the molecular basis of these mechanisms. Since these studies have required mutations, complexation with antibodies, and substitution of detergents in place of lipids, examining the channel under more native conditions is desirable. Solid-state nuclear magnetic resonance (SSNMR) can be used to study the wild-type protein under activating conditions (low pH), at room temperature, and in bacteriomimetic liposomes. In this work, we sought to structurally assign the activated state present in SSNMR experiments. We used a combination of molecular dynamics (MD) simulations, chemical shift prediction algorithms, and Bayesian inference techniques to determine which of the most plausible x-ray structures resolved to date best represents the activated state captured in SSNMR. We first identified specific nuclei with simulated NMR chemical shifts that differed significantly when comparing partially open vs fully open ensembles from MD simulations. The simulated NMR chemical shifts for those specific nuclei were then compared to experimental ones, revealing that the simulation of the partially open state was in good agreement with the SSNMR data. Nuclei that discriminate effectively between partially and fully open states belong to residues spread over the sequence and provide a molecular level description of the conformational change.

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

NMR assignments for the C-terminal domain of human TDP-43.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a 414-residue protein whose aberrant aggregation is implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Intriguingly, TDP-43 has also been shown to functionally oligomerize to carry out physiological functions. TDP-43 also exists in mixed condensates or granules with other proteins (e.g. neuronal or stress granules), and its large C-terminal domain (CTD, residues 267-414) seems responsible for TDP-43 both homo- and heterotypic interactions underlying such diverse functional and pathological aggregation events. A myriad of distinct triggers may drive TDP-43 oligomerization, including interaction partners or changes in pH or salinity. In this Assignment Note, we report the complete backbone and a wealth of side chain chemical shift assignments for the CTD of TDP-43 at pH 4. The assignments presented here provide a solid starting point to study the aggregation pathway of TDP-43 at pH values below those considered physiological but relevant in pathological settings, and to contrast the aggregation behaviour under distinct conditions and in the presence of interacting partners.

NMR studies of lipid regulation of the K+ channel KcsA.

The membrane environment, including specific lipid characteristics, plays important roles in the folding, stability, and gating of the prokaryotic potassium channel KcsA. Here we study the effect of membrane composition on the population of various functional states of KcsA. The spectra provide support for the previous observation of copurifying phospholipids with phosphoglycerol headgroups. Additional, exogenously added anionic lipids do not appear to be required to stabilize the open conductive conformation of KcsA, which was previously thought to be the case. On the contrary, NMR-based binding studies indicate that including anionic lipids in proteoliposomes at acidic pH leads to a weaker potassium ion affinity at the selectivity filter. Since K+ ion loss leads to channel inactivation, these results suggest that anionic lipids promote channel inactivation.

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation.

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets.

Scaled recoupling of chemical shift anisotropies at high magnetic fields under MAS with interspersed C-elements.

The power of chemical shift anisotropy (CSA) measurements for probing structure and dynamics of molecules has been long recognized. NMR pulse sequences that allow measurement of CSA values in an indirect dimension of a protein correlation spectrum have been employed for aliphatic groups, but for practical reasons, carbonyl functional groups have been little studied, despite the fact that carbonyls are expected to give particularly varied and informative CSA values. Specifically, the wide spectral widths of carbonyl tensors make their measurements difficult with typically attainable spectrometer settings. We present here an extended family of experiments that enable the recovery of static CSA lineshapes in an indirect dimension of magic angle spinning (MAS) solid-state NMR experiments, except for various real valued scaling factors. The experiment is suitable for uniformly labeled material, at moderate MAS rates (10 kHz-30 kHz) and at higher magnetic fields (ν0H > 600 MHz). Specifically, the experiments are based on pulse sequence elements from a previous commonly used pulse sequence for CSA measurement, recoupling of chemical shift anisotropy (ROCSA), while modification of scaling factors is achieved by interspersing different blocks of C-elements of the same Cnn 1 cycle. Using experimental conditions similar to the parent ROCSA sequence, a CSA scaling factor between 0 and 0.272 can be obtained, thus allowing a useful practical range of possibilities in experimental conditions for measurement of larger CSA values. Using these blocks, it is also possible to make a constant-time CSA recoupling sequence. The effectiveness of this approach, fROCSA, is shown on model compounds 1-13C-Gly, U-13C,15N-l-His, and microcrystalline U-13C,15N-Ubiquitin.

Probing allosteric coupling in a constitutively open mutant of the ion channel KcsA using solid-state NMR.

Transmembrane allosteric coupling is a feature of many critical biological signaling events. Here we test whether transmembrane allosteric coupling controls the potassium binding affinity of the prototypical potassium channel KcsA in the context of C-type inactivation. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. We tested this mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. The potassium binding affinity in the selectivity filter of this mutant, 81 mM, is about four orders of magnitude weaker than that of wild-type KcsA at neutral pH and is comparable to the value for wild-type KcsA at low pH (pH ≈ 3.5). This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant, the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery.

TmDOTP: An NMR-based thermometer for magic angle spinning NMR experiments.

Solid state NMR is a powerful tool to probe membrane protein structure and dynamics in native lipid membranes. Sample heating during solid state NMR experiments can be caused by magic angle spinning and radio frequency irradiation such heating produces uncertainties in the sample temperature and temperature distribution, which can in turn lead to line broadening and sample deterioration. To measure sample temperatures in real time and to quantify thermal gradients and their dependence on radio frequency irradiation or spinning frequency, we use the chemical shift thermometer TmDOTP, a lanthanide complex. The H6 TmDOTP proton NMR peak has a large chemical shift (-176.3 ppm at 275 K) and it is well resolved from the protein and lipid proton spectrum. Compared to other NMR thermometers (e.g., the proton NMR signal of water), the proton spectrum of TmDOTP, particularly the H6 proton line, exhibits very high thermal sensitivity and resolution. In MAS studies of proteoliposomes we identify two populations of TmDOTP with differing temperatures and dependency on the radio frequency irradiation power. We interpret these populations as arising from the supernatant and the pellet, which is sedimented during sample spinning. In this study, we demonstrate that TmDOTP is an excellent internal standard for monitoring real-time temperatures of biopolymers without changing their properties or obscuring their spectra. Real time temperature calibration is expected to be important for the interpretation of dynamics and other properties of biopolymers.

Stability of nitroxide biradical TOTAPOL in biological samples.

We characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

Identifying coupled clusters of allostery participants through chemical shift perturbations.

Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.

Inactivation in the potassium channel KcsA.

Inactivation, the slow cessation of transmission after activation, is a general feature of potassium channels. It is essential for their function, and malfunctions in inactivation leads to numerous pathologies. The detailed mechanism for the C-type inactivation, distinct from the N-type inactivation, remains an active area of investigation. Crystallography, computational simulations, and NMR have greatly enriched our understanding of the process. Here we review the major hypotheses regarding C-type inactivation, particularly focusing on the key role played by NMR studies of the prokaryotic potassium channel KcsA, which serves as a good model for voltage gated mammalian channels.