Rasmus Linser

Structure Calculation from Unambiguous Long-Range Amide and Methyl 1H−1H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy

Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ∼1.1 Å.

Proton-detected scalar coupling based assignment strategies in MAS solid-state NMR spectroscopy applied to perdeuterated proteins.

Assignment of proteins in MAS (magic angle spinning) solid-state NMR relies so far on correlations among heteronuclei. This strategy is based on well dispersed resonances in the (15)N dimension. In many complex cases like membrane proteins or amyloid fibrils, an additional frequency dimension is desirable in order to spread the amide resonances. We show here that proton detected HNCO, HNCA, and HNCACB type experiments can successfully be implemented in the solid-state. Coherences are sufficiently long lived to allow pulse schemes of a duration greater than 70 ms before incrementation of the first indirect dimension. The achieved resolution is comparable to the resolution obtained in solution-state NMR experiments. We demonstrate the experiments using a triply labeled sample of the SH3 domain of chicken alpha-spectrin, which was re-crystallized in H(2)O/D(2)O using a ratio of 1/9. We employ paramagnetic relaxation enhancement (PRE) using EDTA chelated Cu(II) to enable rapid data acquisition.

Comparison of solid-state dipolar couplings and solution relaxation data provides insight into protein backbone dynamics.

Analyses of solution (15)N relaxation data and solid-state (1)H(N)-(15)N dipolar couplings from a small globular protein, alpha-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns-mus dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns-mus motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of alpha-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.

Probing surface accessibility of proteins using paramagnetic relaxation in solid-state NMR spectroscopy.

Paramagnetic Relaxation Enhancement (PRE) can be used to accelerate NMR data acquisition by reducing the longitudinal proton relaxation time T(1) in the solid state. We show that the presence of paramagnetic compounds in the bulk solvent induces a site-specific relaxation in addition to local dynamics, which is dependent on the surface accessibility of the respective amide proton in the protein. Differentiation between paramagnetic relaxation and dynamics was achieved by a comparison of (1)H T(1) times obtained from microcrystalline protein samples prepared with different concentrations of the Cu(II)(edta) chelate. We find that relaxation can in addition be mediated by hydroxyl groups, which transfer relaxation by their ability to exchange with the quickly relaxing bulk solvent. Furthermore, relaxation seems to be transferred by water molecules which diffuse into the protein structure and yield an efficient difference PRE in flexible regions of the protein. The experiments are demonstrated using a perdeuterated sample of the alpha-spectrin SH3 domain, which was microcrystallized from a buffer containing 90% D(2)O. Deuteration is a prerequisite to avoid spin diffusion which would otherwise compromise site specific resolution.

Direct Observation of Millisecond to Second Motions in Proteins by Dipolar CODEX NMR Spectroscopy

We present a site-resolved study of slow (ms to s) motions in a protein in the solid (microcrystalline) state performed with the use of a modified version of the centerband-only detection of exchange (CODEX) NMR experiment. CODEX was originally based on measuring changes in molecular orientation by means of the chemical shift anisotropy (CSA) tensor, and in our modification, angular reorientations of internuclear vectors are observed. The experiment was applied to the study of slow (15)N-(1)H motions of the SH3 domain of chicken alpha-spectrin. The protein was perdeuterated with partial back-exchange of protons at labile sites. This allowed indirect (proton) detection of (15)N nuclei and thus a significant enhancement of sensitivity. The diluted proton system also made negligible proton-driven spin diffusion between (15)N nuclei, which interferes with the molecular exchange (motion) and hampers the acquisition of dynamic parameters. The experiment has shown that approximately half of the peaks in the 2D (15)N-(1)H correlation spectrum exhibit exchange in a different extent. The correlation time of the slow motion for most peaks is 1 to 3 s. This is the first NMR study of the internal dynamics of proteins in the solid state on the millisecond to second time scale with site-specific spectral resolution that provides both time-scale and geometry information about molecular motions.

Assignment of dynamic regions in biological solids enabled by spin-state selective NMR experiments.

Structural investigations are a prerequisite to understand protein function. Intermediate time scale motional processes (ns-micros) are deleterious for NMR of biological solids and obscure the detection of amide moieties in traditional CP based solid-state NMR approaches as well as in regular scalar coupling based experiments. We show that this obstacle can be overcome by using TROSY type techniques in triple resonance experiments, which enable the assignment of resonances in loop regions of a microcrystalline protein. The presented approach provides an exemplified solution for the analysis of secondary structure elements undergoing slow dynamics that might be particularly crucial for understanding protein function.

Solid-state NMR structure determination from diagonal-compensated, sparsely nonuniform-sampled 4D proton–proton restraints.

We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and 1H back-exchanged proteins. To achieve this, we use homonuclear 1H–1H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal.

Identification of Hydroxyl Protons, Determination of Their Exchange Dynamics, and Characterization of Hydrogen Bonding in a Microcrystallin Protein

Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

A disilene base adduct with a dative Si–Si single bond.

An experimental and theoretical study of the base-stabilized disilene 1 is reported, which forms at low temperatures in the disproportionation reaction of Si2 Cl6 or neo-Si5 Cl12 with equimolar amounts of NMe2 Et. Single-crystal X-ray diffraction and quantum-chemical bonding analysis disclose an unprecedented structure in silicon chemistry featuring a dative Si→Si single bond between two silylene moieties, Me2 EtN→SiCl2 →Si(SiCl3 )2 . The central ambiphilic SiCl2 group is linked by dative bonds to the amine donor and the bis(trichlorosilyl)silylene acceptor, which leads to push-pull stabilization. Based on experimental and theoretical examinations a formation mechanism is presented that involves an autocatalytic reaction of the intermediately formed anion Si(SiCl3 )3 (-) with neo-Si5 Cl12 to yield 1.

Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes

Aggregates formed by amyloidogenic peptides and proteins and reconstituted membrane protein preparations differ significantly in terms of the spectral quality that they display in solid-state NMR experiments. Structural heterogeneity and dynamics can both in principle account for that observation. This perspectives article aims to point out challenges and limitations, but also potential opportunities in the investigation of these systems.