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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. 

Abstract 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, Co₆Se₈(PEt₃)₆. We probed its structural, electronic, and magnetic properties and revealed its detailed electronic structure as valence electrons delocalize over inorganic [Co₆Se₈] core while ligands function as an insulated shell.⁵⁹Co SSNMR measurements on the core and³¹P,¹³C on the ligands show that the neutral Co₆Se₈(PEt₃)₆is 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 [Co₆Se₈(PEt₃)₆]⁺¹is 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. 

ABSTRACT 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. 

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. 

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.