The determination of a representative set of protein structures is a chief aim in structural genomics. Solid-state NMR may have a crucial role in structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems or membrane proteins. Here we present a protein structure determined by solid-state magic-angle-spinning (MAS) NMR. Almost complete (13)C and (15)N resonance assignments for a micro-crystalline preparation of the alpha-spectrin Src-homology 3 (SH3) domain formed the basis for the extraction of a set of distance restraints. These restraints were derived from proton-driven spin diffusion (PDSD) spectra of biosynthetically site-directed, labelled samples obtained from bacteria grown using [1,3-(13)C]glycerol or [2-(13)C]glycerol as carbon sources. This allowed the observation of long-range distance correlations up to approximately 7 A. The calculated global fold of the alpha-spectrin SH3 domain is based on 286 inter-residue (13)C-(13)C and six (15)N-(15)N restraints, all self-consistently obtained by solid-state MAS NMR. This MAS NMR procedure should be widely applicable to small membrane proteins that can be expressed in bacteria.
The small heat shock protein αB-crystallin (αB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric αB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of ~121° between the planes of the β-sandwich formed by α-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with β4 and β8, consistent with a pHdependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with β3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit αB oligomer with tetrahedral symmetry.Small heat shock proteins (sHSPs) help to maintain protein homeostasis by interacting with unfolded, aggregated or misfolded proteins to prevent cell damage [1][2][3] . The ATP-independent chaperone αB-crystallin (αB, 20 kDa, 175 residues) is a paradigm example 4 . αB was originally found in the eye-lens as the B-subunit of α-crystallin, a protein essential for maintaining eyelens transparency. In recent years, the list of biological roles for αB has grown substantially, including involvement in the regulation of the ubiquitin-proteasome pathway as well as AUTHOR CONTRIBUTIONSS.J. contributed to all aspects of the manuscript; P.R. performed solution NMR experiments and helped to write the manuscript; B.B. performed structure calculations; S.M. did solid-state NMR and SAXS measurements as well as data analysis; R.K. contributed to modeling of C-terminal intermolecular interactions; J.R.S. prepared samples; V.A.H. contributed to assignment strategies, was involved in structure calculations and helped write the manuscript; R.E.K. contributed to the interpretation of results and wrote the manuscript; B.J.v.R. contributed to solid-state NMR measurements, discussed the results and helped to write the manuscript; H.O. designed experimental strategies, contributed to the interpretation of results and wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. [6][7][8][9][10][11] . In the brain of patients with Alexander's disease, the insoluble cell fraction contains protein fibers (Rosenthal fibers) coprecipitated with αB phosphorylated at Ser59, whereas unphosphorylated αB remains in the soluble fraction 7 . A missense mutation, R120G, in αB is associated with desmin-related cardiomyopathy 8,9 . The mutations D140N and Q151X are associated with congenital cataracts and myopathy, respectively 10,11 . A decreased concentration of αB in the cerebrospinal fluid was found to be associated with ...
Magic angle spinning (MAS) NMR structure determination is rapidly developing. We demonstrate a method to determine 1 H-13 C distances r CH with high precision from Lee-Goldburg cross-polarization (LG-CP) with fast MAS and continuous LG decoupling on uniformly 13 C-enriched tyrosine‚HCl. The sequence is γ-encoded, and 1 H-13 C spin-pair interactions are predominantly responsible for the polarization transfer while proton spin diffusion is prevented. When the CP amplitudes are set to a sideband of the Hartmann-Hahn match condition, the LG-CP signal builds up in an oscillatory manner, reflecting coherent heteronuclear transfer. Its Fourier transform yields an effective 13 C frequency response that is very sensitive to the surrounding protons. This 13 C spectrum can be reproduced in detail with MAS Floquet simulations of the spin cluster, based on the positions of the nuclei from the neutron diffraction structure. It is symmetric around ω ) 0 and yields two well-resolved maxima. Measurement of CH distances is straightforward, since the separation ∆ω /2π between the maxima for a single 1 H-13 C pair is related to the internuclear distance according to r CH ) a(∆ω/2π) -1/3 , with a ) 25.86 ( 0.01 Å Hz 1/3 . For the 1 H directly bonded to a 13 C, the magnetization is transferred in a short time of ∼100 µs. After this initial rapid transfer period, the COOH, OH, or NH 3 that are not directly bonded to a 13 C transfer magnetization over long distances. This offers an attractive route for collecting long-range distance constraints and for the characterization of intermolecular hydrogen bonding.
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