Chad M. Rienstra

Heteronuclear decoupling in rotating solids

A simple two pulse phase modulation (TPPM) scheme greatly reduces the residual linewidths arising from insufficient proton decoupling power in double resonance magic angle spinning (MAS) experiments. Optimization of pulse lengths and phases in the sequence produces substantial improvements in both the resolution and sensitivity of dilute spins (e.g., 13C) over a broad range of spinning speeds at high magnetic field. The theoretical complications introduced by large homo‐ and heteronuclear interactions among the spins, as well as the amplitude modulation imposed by MAS, are explored analytically and numerically. To our knowledge, this method is the first phase‐switched sequence to exhibit improvement over continuous‐wave (cw) decoupling in a strongly coupled homogeneous spin system undergoing sample spinning.

Homonuclear radio frequency-driven recoupling in rotating solids

We discuss several aspects of homonuclear recoupling and longitudinal exchange using rotor-synchronized spin echo sequences in solid state magic-angle spinning (MAS) experiments. These include the accurate measurement of weak dipole–dipole couplings between rare spins, the behavior of dipolar trajectories in multiple spin environments, and chemical shift correlation spectroscopy via polarization exchange. To describe dipolar trajectories accurately, we adopt an approach to the simulation of these experiments which includes finite pulses and the influence of coherence decay. The latter effect becomes competitive with the strength of weak couplings in many experiments, and a simple empirical approach is outlined for the selection of decay parameters. Dipolar trajectories are shown to be dominated by the largest couplings in multiple spin systems via comparison of two and three interacting spins. Two-dimensional correlation spectroscopy based on dipolar exchange among proximate nuclei is illustrated with a u...

Solid-state NMR structure of a pathogenic fibril of full-length human alpha-synuclein.

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinsons disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register β-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.

De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy

The three-dimensional structure of the chemotactic peptide N-formyl-l-Met-l-Leu-l-Phe-OH was determined by using solid-state NMR (SSNMR). The set of SSNMR data consisted of 16 13C–15N distances and 18 torsion angle constraints (on 10 angles), recorded from uniformly 13C,15N- and 15N-labeled samples. The peptides structure was calculated by means of simulated annealing and a newly developed protocol that ensures that all of conformational space, consistent with the structural constraints, is searched completely. The result is a high-quality structure of a molecule that has thus far not been amenable to single-crystal diffraction studies. The extensions of the SSNMR techniques and computational methods to larger systems appear promising.

Polyphosphate exerts differential effects on blood clotting, depending on polymer size

Polyphosphate, a linear polymer of inorganic phosphate, is secreted by activated platelets and accumulates in many infectious microorganisms. We recently showed that polyphosphate modulates the blood coagulation cascade at 3 steps: it triggers the contact pathway, it accelerates factor V activation, and it enhances fibrin polymerization. We now report that polyphosphate exerts differential effects on blood clotting, depending on polymer length. Very long polymers (≥ 500mers, such as those present in microorganisms) were required for optimal activation of the contact pathway, while shorter polymers (∼ 100mers, similar to the polymer lengths released by platelets) were sufficient to accelerate factor V activation and abrogate the anticoagulant function of the tissue factor pathway inhibitor. Optimal enhancement of fibrin clot turbidity by polyphosphate required ≥ 250mers. Pyrophosphate, which is also secreted by activated platelets, potently blocked polyphosphate-mediated enhancement of fibrin clot structure, suggesting that pyrophosphate is a novel regulator of fibrin function. In conclusion, polyphosphate of the size secreted by platelets is very efficient at accelerating blood clotting reactions but is less efficient at initiating them or at modulating clot structure. Microbial polyphosphate, which is highly procoagulant, may function in host responses to pathogens.

Amphotericin forms an extramembranous and fungicidal sterol sponge

Amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans for over 50 years with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of the first derivatives of amphotericin that kill yeast but not human cells.

Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR

Magic-angle spinning (MAS) solid-state NMR (SSNMR) techniques have emerged in recent years for solving complete structures of uniformly labeled proteins lacking macroscopic order. Strategies used thus far have relied primarily on semiquantitative distance restraints, analogous to the nuclear Overhauser effect (NOE) routinely used in solution NMR. Here, we present a complementary approach for using relative orientations of molecular fragments, determined from dipolar line shapes. Whereas SSNMR distance restraints typically have an uncertainty of ≈1 Å, the tensor-based experiments report on relative vector (pseudobond) angles with precision of a few degrees. By using 3D techniques of this type, vector angle (VEAN) restraints were determined for the majority of the 56-residue B1 immunoglobulin binding domain of protein G [protein GB1 (a total of 47 HN-HN, 49 HN-HC, and 12 HA-HB restraints)]. By using distance restraints alone in the structure calculations, the overall backbone root-mean-square deviation (bbRMSD) was 1.01 ± 0.13 Å (1.52 ± 0.12 Å for all heavy atoms), which improved to 0.49 ± 0.05 Å (1.19 ± 0.07 Å) on the addition of empirical chemical shift [torsion angle likelihood obtained from shift and sequence similarity (TALOS)] restraints. VEAN restraints further improved the ensemble to 0.31 ± 0.06 Å bbRMSD (1.06 ± 0.07 Å); relative to the structure with distances alone, most of the improvement remained (bbRMSD 0.64 ± 0.09 Å; 1.29 ± 0.07 Å) when TALOS restraints were removed before refinement. These results represent significant progress toward atomic-resolution protein structure determination by SSNMR, capabilities that can be applied to a large range of membrane proteins and fibrils, which are often not amenable to solution NMR or x-ray crystallography.

Structured regions of α-Synuclein fibrils include the early-onset Parkinson's disease mutation sites

α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinsons disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD-A30P, E46K and A53T-are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.

High-performance solvent suppression for proton detected solid-state NMR

High-sensitivity proton detected experiments in solid-state NMR have been recently demonstrated in proton diluted proteins as well as fully protonated samples under fast magic-angle spinning. One key element for performing successful proton detection is effective solvent suppression achieved by pulsed field gradients (PFG) and/or saturation pulses. Here we report a high-performance solvent suppression method that attenuates multiple solvent signals simultaneously by more than a factor of 10,000, achieved by an optimized combination of homospoil gradients and supercycled saturation pulses. This method, which we call Multiple Intense Solvent Suppression Intended for Sensitive Spectroscopic Investigation of Protonated Proteins, Instantly (MISSISSIPPI), can be applied without a PFG probe. It opens up new opportunities for two-dimensional heteronuclear correlation spectroscopy of hydrated proteins at natural abundance as well as high-sensitivity and multi-dimensional experimental investigation of protein-solvent interactions.

Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic‐angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three‐dimensional (3D) correlation experiments on a uniformly 13C, 15N‐labeled sample at 750‐MHz 1H frequency. We also present a four‐dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.