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

DNP Enhanced Frequency-Selective TEDOR Experiments in Bacteriorhodopsin

We describe a new approach to multiple [superscript 13]C–[superscript 15]N distance measurements in uniformly labeled solids, frequency-selective (FS) TEDOR. The method shares features with FS-REDOR and ZF- and BASE-TEDOR, which also provide quantitative [superscript 15]N–[superscript 13]C spectral assignments and distance measurements in U-[[superscript 13]C,[superscript 15]N] samples. To demonstrate the validity of the FS-TEDOR sequence, we measured distances in [U-[superscript 13]C,15N]-asparagine which are in good agreement with other methods. In addition, we integrate high frequency dynamic nuclear polarization (DNP) into the experimental protocol and use FS-TEDOR to record a resolved correlation spectrum of the Arg-[superscript 13]Cγ–[superscript 15]Nε region in [U-[superscript 13]C,15N]-bacteriorhodopsin. We resolve six of the seven cross-peaks expected based on the primary sequence of this membrane protein.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-001960)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-002804)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-001035)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-002026

Resolution and Polarization Distribution in Cryogenic DNP/MAS Experiments

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz [superscript 1]H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between [superscript 1]H–[superscript 1]H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026)National Institutes of Health (U.S.) (Grant EB001965)National Institutes of Health (U.S.) (Grant EB004866)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Subunit Structure of Gas Vesicles: A MALDI-TOF Mass Spectrometry Study

Many aquatic microorganisms use gas vesicles to regulate their depth in the water column. The molecular basis for the novel physical properties of these floatation organelles remains mysterious due to the inapplicability of either solution or single crystal structural methods. In the present study, some folding constraints for the ∼7-kDa GvpA building blocks of the vesicles are established via matrix-assisted laser desorption ionization time-of-flight mass spectrometry studies of intact and proteolyzed vesicles from the cyanobacterium Anabaena flos-aquae and the archaea Halobacterium salinarum. The spectra of undigested vesicles show no evidence of posttranslational modification of the GvpA. The extent of carboxypeptidase digestion shows that the alanine rich C-terminal pentapeptide of GvpA is exposed to the surface in both organisms. The bonds that are cleaved by Trypsin and GluC are exclusively in the extended N-terminus of the Anabaena flos-aquae protein and in the extended C-terminus of the Halobacterium salinarum protein. All the potentially cleavable peptide bonds in the central, highly conserved portion of the protein appear to be shielded from protease attack in spite of the fact that some of the corresponding side chains are almost certainly exposed to the aqueous medium

Gas Vesicles across Kingdoms: A Comparative Solid-State Nuclear Magnetic Resonance Study

The buoyancy organelles of aquatic microorganisms have to meet stringent specifications: allowing gases to equilibrate freely across the proteinaceous shell, preventing the condensation of water vapor inside the hollow cavity and resisting collapse under hydrostatic pressures that vary with column depth. These properties are provided by the 7- to 8-kDa gas vesicle protein A (GvpA), repeats of which form all but small, specialized portions of the shell. Magic angle spinning nuclear magnetic resonance is uniquely capable of providing high-resolution information on the fold and assembly of GvpA. Here we compare results for the gas vesicles of the haloarchaea Halobacterium salinarum with those obtained previously for the cyanobacterium Anabaena flos-aquae. The data suggest that the two organisms follow similar strategies for avoiding water condensation. On the other hand, in its relatively shallow habitat, H. salinarum is able to avoid collapse with a less costly GvpA fold than is adopted by A. flos-aquae.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