Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-[superscript 13]C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-[superscript 13]C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001035)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001960)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-002026

Solid-state NMR evidence for inequivalent GvpA subunits in gas vesicles

Gas vesicles are organelles that provide buoyancy to the aquatic microorganisms that harbor them. The gas vesicle shell consists almost exclusively of the hydrophobic 70-residue gas vesicle protein A, arranged in an ordered array. Solid-state NMR spectra of intact collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae show duplication of certain gas vesicle protein A resonances, indicating that specific sites experience at least two different local environments. Interpretation of these results in terms of an asymmetric dimer repeat unit can reconcile otherwise conflicting features of the primary, secondary, tertiary, and quaternary structures of the gas vesicle protein. In particular, the asymmetric dimer can explain how the hydrogen bonds in the β-sheet portion of the molecule can be oriented optimally for strength while promoting stabilizing aromatic and electrostatic side-chain interactions among highly conserved residues and creating a large hydrophobic surface suitable for preventing water condensation inside the vesicle.National Institutes of Health (U.S.) (Grant EB002175)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026

Site-Resolved Measurement of Microsecond-to-Millisecond Conformational-Exchange Processes in Proteins by Solid-State NMR Spectroscopy

We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr–Purcell–Meiboom–Gill relaxation-dispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers.ISSN:0002-7863ISSN:1520-512

Probing Transient Conformational States of Proteins by Solid-State R 11 Relaxation-Dispersion NMR Spec- troscopy

International audienceSolution-state NMR spectroscopic techniques, and in par-ticular so-called relaxation-dispersion (RD) NMR approaches, have proven very successful for studying ms–ms motion and characterizing the exchanging short-lived con-formations. [1] RD NMR techniques exploit the effect of conformational-exchange processes on line broadening, that is, on the relaxation rates of nuclear spin coherence (R 2 , R 11). By quantifying spin relaxation rates in the presence of a variable radiofrequency (rf) field, RD approaches provide information about relative populations and exchange rates, as well as chemical shifts of short-lived conformational states, and thus about local structure. [1, 2] In the case of very large assemblies or insoluble aggregates, where solution-state NMR is severely challenged, magic-angle-spinning solid-state NMR (MAS ssNMR) is rapidly emerging as a tool for the study of structure and dynamics. However, the character-ization of conformational-exchange dynamics in the solid state remains challenging. Herein, we show a ssNMR approach in which amide-15 N R 11 RD data, that is, the rate of coherence decay under 15 N spin-lock fields of variable field strengths, are quantitatively analyzed and interpreted in terms of conformational dynamics, providing insight into short-lived states in terms of chemical shifts and bond vector orientations. We investigate the robustness of this approach by studying a conformational flip in crystalline ubiquitin. Conformational fluctuations between different states expose a given nuclear spin in a protein to different local environments, which are characterized by different bond geometries. A simple case, exchange between two states, is shown in Figure 1 a. As a result of conformational-exchange dynamics, a given spin will experience a fluctuation of its chemical shift (CS), as well as of its bond vector orientations and, thus, dipolar coupling interactions with neighboring spins and CS anisotropies (CSA). In solution-state NMR, dipolar coupling and CSA interactions are averaged to zero by Brownian movement (molecular tumbling). Consequently, only fluctuations of the isotropic CS are relevant when considering conformational dynamics on the microsecond to millisecond timescale. R 11 RD experiments in solution can thus only pick up conformational dynamics if they involve a change in the isotropic CS. The relevant theory is well established, and the effects of exchange can be described by the Bloch–McConnell equations. [3

Site-Resolved Measurement of Microsecond-to-Millisecond Conformational-Exchange Processes in Proteins by Solid-State NMR Spectroscopy

We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr–Purcell–Meiboom–Gill relaxation-dispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers

Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

International audienceAtomic-resolution structure determination is crucial for understanding protein function.Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EMdoes not routinely give access to atomic-level structural data, and, generally, NMR structuredetermination is restricted to small (<30 kDa) proteins. We introduce an integrated structuredetermination approach that simultaneously uses NMR and EM data to overcome the limitsof each of these methods. The approach enables structure determination of the 468 kDalarge dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combiningsecondary-structure information obtained from near-complete magic-angle-spinning NMRassignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILVmethyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds currentstandards of NMR and EM structure determination in terms of molecular weight and pre-cision. Importantly, the approach is successful even in cases where only medium-resolutioncryo-EM data are availabl

Solid-State NMR Characterization of Gas Vesicle Structure

Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil-α-β-β-α-coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface

Site-Resolved Measurement of Microsecond-to-Millisecond Conformational-Exchange Processes in Proteins by Solid-State NMR Spectroscopy

We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr–Purcell–Meiboom–Gill relaxation-dispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers.ISSN:0002-7863ISSN:1520-512