Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving ¹³C and ¹⁵N spins have been developed for this purpose, ¹H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular ¹H-¹³C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved ¹H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR, simplifies the ¹H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced ¹H chemical shift resolution allows the assignment of overlapping H and H resonances in Ser, identifies the ¹H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

Solid-state NMR and functional studies on proteorhodopsin

Proteins of the proteorhodopsin (PR) family are found abundantly in many marine bacteria in the photic zone of the oceans. They are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump and thus could form a potential source of energy. The pK_a of the primary proton acceptor is close to the pH of seawater which could also indicate a regulatory role. Here, we review and summarize our own recent findings in the context of known data and present some new results. Proton transfer in vitro by PR is shown by a fluorescence assay which confirms a pH dependent vectoriality. Previously reported low diffracting 2D crystal preparations of PR are assessed for their use for solid-state NMR by two dimensional ¹³C-¹³C DARR spectra. ¹⁵N-¹H HETCOR MAS NMR experiments show bound water in the vicinity of the protonated Schiff base which could play a role in proton transfer. The effect of highly conserved H75 onto the properties of the chromophore has been investigated by single site mutations. They do show a pronounced effect onto the optical absorption maximum and the pK_a of the proton acceptor but have only a small effect onto the ¹⁵N chemical shifts of the protonated Schiff base.     

Probing water accessibility in HET-s(218-289) amyloid fibrils by solid-state NMR

Despite the importance of protein fibrils in the context of conformational diseases, information on their structure is still sparse. Hydrogen/deuterium exchange measurements of backbone amide protons allow the identification hydrogen-bonding patterns and reveal pertinent information on the amyloid β-sheet architecture. However, they provide only little information on the identity of residues exposed to solvent or buried inside the fibril core. NMR spectroscopy is a potent method for identifying solvent-accessible residues in proteins via observation of polarization transfer between chemically exchanging side-chain protons and water protons. We show here that the combined use of highly deuterated samples and fast magic-angle spinning greatly attenuates unwanted spin diffusion and allows identification of polarization exchange with the solvent in a site-specific manner. We apply this measurement protocol to HET-s(218-289) prion fibrils under different conditions (including physiological pH, where protofibrils assemble together into thicker fibrils) and demonstrate that each protofibril of HET-s(218-289), is surrounded by water, thus excluding the existence of extended dry interfibril contacts. We also show that exchangeable side-chain protons inside the hydrophobic core of HET-s(218-289) do not exchange over time intervals of weeks to months. The experiments proposed in this study can provide insight into the detailed structural features of amyloid fibrils in general.     

NMR crystallography: The effect of deuteration on high resolution C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the ¹³C linewidths of U-¹³C, ¹⁵N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in ²H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by < 0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ₁, and Cγ₂ Ile ¹³C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured ¹³C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. ¹H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power ¹H decoupling is required.     

Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16-A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2-G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state ¹H/¹⁵N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly.     

Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein

Fibrils from the Parkinson's-disease-related A53T mutant of α-synuclein were investigated by solid-state NMR spectroscopy, electron microscopy, and atomic force microscopy. Sequential solid-state NMR resonance assignments were obtained for a large fraction of the fibril core. Experiments conducted above and below the freezing point suggest that the fibrils contain regions with increased mobility and structural elements different from β-strand character, in addition to the rigid β-sheet-rich core region. As in earlier studies on wild-type α-synuclein, the C-terminus was found to be flexible and unfolded, whereas the main core region was highly rigid and rich in β-sheets. Compared to fibrils from wild-type α-synuclein, the well-ordered β-sheet region extends to at least L38 and L100. These results demonstrate that a disease-related mutant of α-synuclein differs in both aggregation kinetics and fibril structure.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

Proteorhodopsin: Characterisation of 2D crystals by electron microscopy and solid state NMR

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in γ-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by ¹⁵N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.