J. Am. Chem. Soc., 2010, 132 (16), pp 5538–5539
Breaking the T1 Constraint for Quantitative Measurement in Magic Angle Spinning Solid-State NMR Spectroscopy
Guangjin Hou, Shangwu Ding, Limin Zhang and Feng Deng
Quantitative solid-state NMR experimental schemes that break the conventional T1 constraint are described. The combination of broad-band homonuclear recoupling techniques and the conventional single pulse or cross-polarization (CP) schemes (referred as QUSP or QUCP) render the long T1 of low-? spins no longer a constraint for obtaining quantitative NMR spectra. During the mixing time when dipolar recoupling occurs, the nonuniformly CP enhanced or recovered spin magnetization is redistributed under the reintroduced homonuclear dipole-dipole interactions so that uniformly enhanced or recovered magnetization is achieved when the system reaches the quasi-equilibrium state. It is shown that quantitative NMR spectra can be obtained for the recycle delays substantially shorter than the conventionally required 5T1. In addition, the high efficiency gain can be achieved in QUSP and QUCP experiments with a relatively short recycle delay.
J. Am. Chem. Soc., 2010, 132 (16), pp 5546–5547
Detection of Transient Interchain Interactions in the Intrinsically Disordered Protein a-Synuclein by NMR Paramagnetic Relaxation Enhancement
Kuen-Phon Wu and Jean Baum
NMR paramagnetic relaxation enhancement experiments were applied to the intrinsically disordered protein a-synuclein, the primary protein in Parkinson's disease, to directly characterize transient intermolecular complexes at neutral and low pH. At neutral pH, we observed weak N- to C-terminal interchain contacts driven by electrostatic interactions, while at low pH, the C- to C-terminal interchain interactions are significantly stronger and driven by hydrophobic contacts. Characterization of these first interchain interactions will provide fundamental insight into the mechanism of amyloid formation.
J. Am. Chem. Soc., 2010, 132 (16), pp 5556–5557
Fibrillar vs Crystalline Full-Length ß-2-Microglobulin Studied by High-Resolution Solid-State NMR Spectroscopy
Emeline Barbet-Massin†, Stefano Ricagno‡§, Józef R. Lewandowski†, Sofia Giorgetti§, Vittorio Bellotti‡§, Martino Bolognesi, Lyndon Emsley† and Guido Pintacuda*†
Elucidating the fine structure of amyloid fibrils as well as understanding their processes of nucleation and growth remains a difficult yet essential challenge, directly linked to our current poor insight into protein misfolding and aggregation diseases. Here we consider ß-2-microglobulin (ß2m), the MHC-1 light chain component responsible for dialysis-related amyloidosis, which can give rise to amyloid fibrils in vitro under various experimental conditions, including low and neutral pH. We have used solid-state NMR to probe the structural features of fibrils formed by full-length ß2m (99 residues) at pH 2.5 and pH 7.4. A close comparison of 2D 13C-13C and 15N-13C correlation experiments performed on ß2m, in both the crystalline and fibrillar states, suggests that, in spite of structural changes affecting the protein loops linking the protein ß-strands, the protein chain retains a substantial share of its native secondary structure in the fibril assembly. Moreover, variations in the chemical shifts of the key Pro32 residue suggest the involvement of a cis-trans isomerization in the process of ß2m fibril formation. Lastly, the analogy of the spectra recorded on ß2m fibrils grown at different pH values hints at a conserved architecture of the amyloid species thus obtained.
J. Am. Chem. Soc., 2010, 132 (16), pp 5558–5559
Ultrafast MAS Solid-State NMR Permits Extensive 13C and 1H Detection in Paramagnetic Metalloproteins
Ivano Bertini, Lyndon Emsley§, Moreno Lelli, Claudio Luchinat, Jiafei Mao and Guido Pintacuda
We show here that by combining tailored approaches based on ultrafast (60 kHz) MAS on the CoII-replaced catalytic domain of matrix metalloproteinase 12 (CoMMP-12) we can observe and assign, in a highly paramagnetic protein in the solid state, 13C and even 1H resonances from the residues coordinating the metal center. In addition, by exploiting the enhanced relaxation caused by the paramagnetic center, and the low power irradiation enabled by the fast MAS, this can be achieved in remarkably short times and at very high field (21.2 T), with only less than 1 mg of sample. Furthermore, using the known crystal structure of the compound, we are able to distinguish and measure pseudocontact (PCS) contributions to the shifts up to the coordinating ligands and to unveil structural information.
J. Am. Chem. Soc., 2010, 132 (16), pp 5672–5676
NMR-Based Structural Modeling of Graphite Oxide Using Multidimensional 13C Solid-State NMR and ab Initio Chemical Shift Calculations
Leah B. Casabianca†, Medhat A. Shaibat†, Weiwei W. Cai‡, Sungjin Park‡, Richard Piner‡, Rodney S. Ruoff‡ and Yoshitaka Ishii†
Chemically modified graphenes and other graphite-based materials have attracted growing interest for their unique potential as lightweight electronic and structural nanomaterials. It is an important challenge to construct structural models of noncrystalline graphite-based materials on the basis of NMR or other spectroscopic data. To address this challenge, a solid-state NMR (SSNMR)-based structural modeling approach is presented on graphite oxide (GO), which is a prominent precursor and interesting benchmark system of modified graphene. An experimental 2D 13C double-quantum/single-quantum correlation SSNMR spectrum of 13C-labeled GO was compared with spectra simulated for different structural models using ab initio geometry optimization and chemical shift calculations. The results show that the spectral features of the GO sample are best reproduced by a geometry-optimized structural model that is based on the Lerf-Klinowski model (Lerf, A. et al. Phys. Chem. B 1998, 102, 4477); this model is composed of interconnected sp2, 1,2-epoxide, and COH carbons. This study also convincingly excludes the possibility of other previously proposed models, including the highly oxidized structures involving 1,3-epoxide carbons (Szabo, I. et al. Chem. Mater. 2006, 18, 2740). 13C chemical shift anisotropy (CSA) patterns measured by a 2D 13C CSA/isotropic shift correlation SSNMR were well reproduced by the chemical shift tensor obtained by the ab initio calculation for the former model. The approach presented here is likely to be applicable to other chemically modified graphenes and graphite-based systems.
J. Am. Chem. Soc., 2010, 132 (16), pp 5779–5788
Proton-Evolved Local-Field Solid-State NMR Studies of Cytochrome b5 Embedded in Bicelles, Revealing both Structural and Dynamical Information
Ronald Soong†, Pieter E. S. Smith†, Jiadi Xu†, Kazutoshi Yamamoto†, Sang-Choul Im‡, Lucy Waskell‡ and Ayyalusamy Ramamoorthy*†
Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented two-dimensional proton-evolved local-field (2D PELF) pulse sequences using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically aligned bicelles containing uniformly 15N-labeled cytochrome b5 are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF-based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.
J. Am. Chem. Soc., 2010, 132 (16), pp 5803–5811
Changes in Transmembrane Helix Alignment by Arginine Residues Revealed by Solid-State NMR Experiments and Coarse-Grained MD Simulations
Vitaly V. Vostrikov‡†, Benjamin A. Hall§†, Denise V. Greathouse‡, Roger E. Koeppe, II*‡ and Mark S. P. Sansom*§
Independent experimental and computational approaches show agreement concerning arginine/membrane interactions when a single arginine is introduced at selected positions within the membrane-spanning region of acetyl-GGALW5LALALAL12AL14ALALW19LAGA-ethanolamide, designated GWALP23. Peptide sequence isomers having Arg in position 12 or position 14 display markedly different behaviors, as deduced by both solid-state NMR experiments and coarse-grained molecular dynamics (CG-MD) simulations. With respect to the membrane normal of DOPC or DPPC lipid bilayer membranes, GWALP23-R14 shows one major state whose apparent average tilt is 10° greater than that of GWALP23. The presence of R14 furthermore induces bilayer thinning and peptide displacement to "lift" the charged guanidinium toward the bilayer surface. By contrast, GWALP23-R12 exhibits multiple states that are in slow exchange on the NMR time scale, with CG-MD simulations indicating two distinct positions with different screw rotation angles in the membrane, along with an increased tendency to exit the lipid bilayer.