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.     

Quantitative analysis of protein backbone dynamics in microcrystalline ubiquitin by solid-state NMR spectroscopy

Characterization of protein dynamics by solid-state NMR spectroscopy requires robust and accurate measurement protocols, which are not yet fully developed. In this study, we investigate the backbone dynamics of microcrystalline ubiquitin using different approaches. A rotational-echo double-resonance type (REDOR-type) methodology allows one to accurately measure (1)H-(15)N order parameters in highly deuterated samples. We show that the systematic errors in the REDOR experiment are as low as 1% or even less, giving access to accurate data for the amplitudes of backbone mobility. Combining such dipolar-coupling-derived order parameters with autocorrelated and cross-correlated (15)N relaxation rates, we are able to quantitate amplitudes and correlation times of backbone dynamics on picosecond and nanosecond time scales in a residue-resolved manner. While the mobility on picosecond time scales appears to have rather uniform amplitude throughout the protein, we unambiguously identify and quantitate nanosecond mobility with order parameters S(2) as low as 0.8 in some regions of the protein, where nanosecond dynamics has also been revealed in solution state. The methodology used here, a combination of accurate dipolar-coupling measurements and different relaxation parameters, yields details about dynamics on different time scales and can be applied to solid protein samples such as amyloid fibrils or membrane proteins.     

Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis

Magic-angle spinning solid-state NMR (SSNMR) studies of the beta1 immunoglobulin binding domain of protein G (GB1) are presented. Chemical shift correlation spectra at 11.7 T (500 MHz 1H frequency) were employed to identify signals specific to each amino acid residue type and to establish backbone connectivities. High sensitivity and resolution facilitated the detection and assignment of every 15N and 13C site, including the N-terminal (M1) 15NH3, the C-terminal (E56) 13C', and side-chain resonances from residues exhibiting fast-limit conformational exchange near room temperature. The assigned spectra lend novel insight into the structure and dynamics of microcrystalline GB1. Secondary isotropic chemical shifts report on conformation, enabling a detailed comparison of the microcrystalline state with the conformation of single crystals and the protein in solution; the consistency of backbone conformation in these three preparations is the best among proteins studied so far. Signal intensities and line widths vary as a function of amino acid position and temperature. High-resolution spectra are observed near room temperature (280 K) and at <180 K, whereas resolution and sensitivity greatly degrade substantially near 210 K; the magnitude of this effect is greatest among the side chains of residues at the intermolecular interface of the microcrystal lattice, which we attribute to intermediate-rate translational diffusion of solvent molecules near the glass transition. These features of GB1 will enable its use as an excellent model protein not only for SSNMR methods development but also for fundamental studies of protein thermodynamics in the solid state.     

Order parameters based on (13)C(1)H, (13)C(1)H(2) and (13)C(1)H(3) heteronuclear dipolar powder patterns: a comparison of MAS-based solid-state NMR sequences

Order parameters describing conformational exchange processes on the nanosecond to microsecond timescale can be obtained from powder patterns in solid-state NMR (SSNMR) experiments. Extensions of these experiments to magic-angle spinning (MAS) based high-resolution experiments have been demonstrated, which show a great promise for site-specific probes of biopolymers. In this study, we present a detailed comparison of two pulse sequences, transverse Manfield-Rhim-Elleman-Vaughn (T-MREV) and Lee-Goldburg cross-polarization (LGCP), using experimental and simulation tools to explore their utility in the study of order parameters. We discuss systematic errors due to passively coupled (13)C or (1)H nuclei, as well as due to B(1) inhomogeneity. Both pulse sequences can provide quantitative measurements of the order parameter, but the LGCP experiment is capable of greater accuracy provided that the B(1) field is highly homogeneous. The T-MREV experiment is far better compensated for B(1) inhomogeneity, and it also performs better in situations with limited signal.     

Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.     

Determinations of 15N chemical shift anisotropy magnitudes in a uniformly 15N,13C-labeled microcrystalline protein by three-dimensional magic-angle spinning nuclear magnetic resonance spectroscopy

Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics. Experimental determinations of 15N CSA tensors in biologically relevant molecules have typically been performed by NMR relaxation studies in solution, goniometric analysis of single-crystal spectra, or slow magic-angle spinning (MAS) NMR experiments of microcrystalline samples. Here we present measurements of 15N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR. Isotropic 15N, 13C alpha, and 13C' chemical shifts in two dimensions resolve site-specific backbone amide recoupled CSA line shapes in the third dimension. Application of the experiments to the 56-residue beta1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of 15N tensors at 51 of the 55 backbone amide sites, for which 15N-13C alpha and/or 15N-13C' cross-peaks were resolved in the two-dimensional experiment. For 37 15N signals, both intra- and interresidue correlations were resolved, enabling direct comparison of two experimental data sets to enhance measurement precision. Systematic variations between beta-sheet and alpha-helix residues are observed; the average value for the anisotropy parameter, delta (delta = delta(zz) - delta(iso)), for alpha-helical residues is 6 ppm greater than that for the beta-sheet residues. The results show a variation in delta of 15N amide backbone sites between -77 and -115 ppm, with an average value of -103.5 ppm. Some sites (e.g., G41) display smaller anisotropy due to backbone dynamics. In contrast, we observe an unusually large 15N tensor for K50, a residue that has an atypical, positive value for the backbone phi torsion angle. To our knowledge, this is the most complete experimental analysis of 15N CSA magnitude to date in a solid protein. The availability of previous high-resolution crystal and solution NMR structures, as well as detailed solid-state NMR studies, will enhance the value of these measurements as a benchmark for the development of ab initio calculations of amide 15N shielding tensor magnitudes.     

The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.     

Backbone dynamics in the DNA HhaI protein binding site

The dynamics of the phosphodiester backbone in the [5'-GCGC-3'] 2 moiety of the DNA oligomer [d(G 1A 2T 3A 4 G 5 C 6 G 7 C 8T 9A 10T 11C 12)] 2 are studied using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs nonstereospecifically deuterated on the 5' methylene group of nucleotides within the [5'-GCGC-3'] 2 moiety indicated that all of these positions are structurally flexible. Previous work has shown that methylation reduces the amplitude of motion in the phosphodiester backbone and furanose ring of the same DNA, and our observations indicate that methylation perturbs backbone dynamics through not only a loss of mobility but also a change of direction of motion. These NMR data indicate that the [5'-GCGC-3'] 2 moiety is dynamic, with the largest amplitude motions occurring nearest the methylation site. The change of orientation of this moiety in DNA upon methylation may make the molecule less amenable to binding to the HhaI endonuclease.     

Progress in 13C and 1H solid-state nuclear magnetic resonance for paramagnetic systems under very fast magic angle spinning

High-resolution solid-state NMR (SSNMR) of paramagnetic systems has been largely unexplored because of various technical difficulties due to large hyperfine shifts, which have limited the success of previous studies through depressed sensitivity/resolution and lack of suitable assignment methods. Our group recently introduced an approach using "very fast" magic angle spinning (VFMAS) for SSNMR of paramagnetic systems, which opened an avenue toward routine analyses of small paramagnetic systems by (13)C and (1)H SSNMR [Y. Ishii et al., J. Am. Chem. Soc. 125, 3438 (2003); N. P. Wickramasinghe et al., ibid. 127, 5796 (2005)]. In this review, we discuss our recent progress in establishing this approach, which offers solutions to a series of problems associated with large hyperfine shifts. First, we demonstrate that MAS at a spinning speed of 20 kHz or higher greatly improves sensitivity and resolution in both (1)H and (13)C SSNMR for paramagnetic systems such as Cu(II)(DL-alanine)(2)H(2)O (Cu(DL-Ala)(2)) and Mn(acac)(3), for which the spectral dispersions due to (1)H hyperfine shifts reach 200 and 700 ppm, respectively. Then, we introduce polarization transfer methods from (1)H spins to (13)C spins with high-power cross polarization and dipolar insensitive nuclei enhanced by polarization transfer (INEPT) in order to attain further sensitivity enhancement and to correlate (1)H and (13)C spins in two-dimensional (2D) SSNMR for the paramagnetic systems. Comparison of (13)C VFMAS SSNMR spectra with (13)C solution NMR spectra revealed superior sensitivity in SSNMR for Cu(DL-Ala)(2), Cu(Gly)(2), and V(acac)(3). We discuss signal assignment methods using one-dimensional (1D) (13)C SSNMR (13)C-(1)H rotational echo double resonance (REDOR) and dipolar INEPT methods and 2D (13)C(1)H correlation SSNMR under VFMAS, which yield reliable assignments of (1)H and (13)C resonances for Cu(Ala-Thr). Based on the excellent sensitivity/resolution and signal assignments attained in the VFMAS approach, we discuss methods of elucidating multiple distance constraints in unlabeled paramagnetic systems by combing simple measurements of (13)C T(1) values and anisotropic hyperfine shifts. Comparison of experimental (13)C hyperfine shifts and ab initio calculated shifts for alpha- and beta-forms of Cu(8-quinolinol)(2) demonstrates that (13)C hyperfine shifts are parameters exceptionally sensitive to small structural difference between the two polymorphs. Finally, we discuss sensitivity enhancement with paramagnetic ion doping in (13)C SSNMR of nonparamagnetic proteins in microcrystals. Fast recycling with exceptionally short recycle delays matched to short (1)H T(1) of approximately 60 ms in the presence of Cu(II) doping accelerated 1D (13)C SSNMR for ubiquitin and lysozyme by a factor of 7.3-8.4 under fast MAS at a spinning speed of 40 kHz. It is likely that the VFMAS approach and use of paramagnetic interactions are applicable to a variety of paramagnetic systems and nonparamagnetic biomolecules.     

Solid-state nuclear magnetic resonance spectroscopy studies of furanose ring dynamics in the DNA HhaI binding site

The dynamics of the furanose rings in the GCGC moiety of the DNA oligomer [d(G 1A 2T 3A 4 G 5 C 6 G 7 C 8T 9A 10T 11C 12)] 2 are studied by using deuterium solid-state NMR (SSNMR). SSNMR spectra obtained from DNAs selectively deuterated on the furanose rings of nucleotides within the 5'-GCGC-3' moiety indicated that all of these positions are structurally flexible. The furanose ring within the deoxycytidine that is the methylation target displays the largest-amplitude structural changes according to the observed deuterium NMR line shapes, whereas the furanose rings of nucleotides more remote from the methylation site have less-mobile furanose rings (i.e., with puckering amplitudes < 0.3 A). Previous work has shown that methylation reduces the amplitude of motion in the phosphodiester backbone of the same DNA, and our observations indicate that methylation perturbs backbone dynamics through the furanose ring. These NMR data indicate that the 5'-GCGC-3' is dynamic, with the largest-amplitude motions occurring nearest the methylation site. The inherent flexibility of this moiety in DNA makes the molecule more amenable to the large-amplitude structural rearrangements that must occur when the DNA binds to the HhaI methyltransferase.     

Supramolecular assembly of dendritic polymers elucidated by 1H and 13C solid-state MAS NMR spectroscopy

Advanced solid-state NMR methods under fast magic-angle spinning (MAS) are used to study the structure and dynamics of large supramolecular systems, which consist of a polymer backbone with dendritic side groups and self-assemble into a columnar structure. The NMR experiments are performed on as-synthesized samples, i.e., no isotopic enrichment is required. The analysis of (1)H NMR chemical-shift effects as well as dipolar (1)H-(1)H or (1)H-(13)C couplings provide site-specific insight into the local structure and the segmental dynamics, in particular, of phenyl rings and -CH(2)O- linking units within the dendrons. Relative changes of (1)H chemical shifts (of up to -3 ppm) serve as distance constraints and allow protons to be positioned relative to aromatic rings. Together with dipolar spinning sideband patterns, pi-pi packing phenomena and local order parameters (showing variations between 30% and 100%) are selectively and precisely determined, enabling the identification of the dendron cores as the structure-directing moieties within the supramolecular architecture. The study is carried out over a representative selection of systems which reflect characteristic differences, such as different polymer backbones, sizes of dendritic side groups, or length and flexibility of linking units. While the polymer backbone is found to have virtually no effect on the overall structure and properties, the systems are sensitively affected by changing the generation or the linkage of the dendrons. The results help to understand the self-assembly process of dendritic moieties and aid the chemical design of self-organizing molecular structures.     

Assignment of the backbone resonances for microcrystalline ubiquitin

Site-specific assignments for the solid-state NMR spectra of uniformly 13C,15N-enriched ubiquitin are described. The assignments are derived from three three-dimensional 15N-13C-13C correlation spectra collected at 400 MHz on microcrystalline material. A few residues (the loop near Threonine 9 and the C-terminal fragment) were missing and correspond to regions previously reported to be mobile on the basis of X-ray crystallography and solution NMR studies. A few additional sites exhibit shifts that differ from previously reported solution NMR assignments. Nonetheless, these de novo assignments indicate close agreement between the chemical shifts observed in solution and those in microcrystalline or precipitated solids. The methods utilized are likely to be generally applicable for other noncrystalline, nonsoluble proteins.     

Crystal polymorphism of protein GB1 examined by solid-state NMR spectroscopy and X-ray diffraction

The study of micro- or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR) gives atomic-resolution insight into structure in cases when single crystals cannot be obtained for diffraction studies. Subtle differences in the local chemical environment around the protein, including the characteristics of the cosolvent and the buffer, determine whether a protein will form single crystals. The impact of these small changes in formulation is also evident in the SSNMR spectra; however, the changes lead only to correspondingly subtle changes in the spectra. Here, we demonstrate that several formulations of GB1 microcrystals yield very high quality SSNMR spectra, although only a subset of conditions enable growth of single crystals. We have characterized these polymorphs by X-ray powder diffraction and assigned the SSNMR spectra. Assignments of the 13C and 15N SSNMR chemical shifts confirm that the backbone structure is conserved, indicative of a common protein fold, but side chain chemical shifts are changed on the surface of the protein, in a manner dependent upon crystal packing and electrostatic interactions with salt in the mother liquor. Our results demonstrate the ability of SSNMR to reveal minor structural differences among crystal polymorphs. This ability has potential practical utility for studying the formulation chemistry of industrial and therapeutic proteins, as well as for deriving fundamental insights into the phenomenon of single-crystal growth.     

Natural abundance solid-state 67Zn NMR characterization of microporous zinc phosphites and zinc phosphates at ultrahigh magnetic field

Zinc-phosphite and -phosphate based microporous materials are crystalline open framework materials with potential industrial applications. Although (31)P MAS NMR has been used for characterization of these materials, the local environments around zinc centres have never been directly probed by solid-state NMR due to the many unfavourable NMR characteristics of (67)Zn. In this work, we have characterized the local structure around the Zn centres in several representative microporous zinc phosphites and zinc phosphates by acquiring natural abundance (67)Zn solid-state NMR spectra at ultrahigh magnetic field of 21.1 T. The observed line-shapes are mainly determined by the second order quadrupolar interaction. The NMR tensor parameters were extracted from the spectra and are related to the local geometry around the Zn centre. Computational study of the electric field gradient (EFG) tensor at Zn was performed using hybrid density functional theory (DFT) calculations at B3LYP level of theory on model clusters. The calculations using Projector Augmented-Wave (PAW) method were also carried out with the CASTEP code wherever it was possible. The work has shown that it is possible to study Zn environments in porous materials which often have very low Zn concentration by natural abundance (67)Zn SSNMR at very high magnetic fields.     

Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins.

The effects of internal motions on residual dipolar NMR couplings of proteins partially aligned in a liquid-crystalline environment are analyzed using a 10 ns molecular dynamics (MD) computer simulation of ubiquitin. For a set of alignment tensors with different orientations and rhombicities, MD-averaged dipolar couplings are determined and subsequently interpreted for different scenarios in terms of effective alignment tensors, average orientations of dipolar vectors, and intramolecular reorientational vector distributions. Analytical relationships are derived that reflect similarities and differences between motional scaling of dipolar couplings and scaling of dipolar relaxation data (NMR order parameters). Application of the self-consistent procedure presented here to dipolar coupling measurements of biomolecules aligned in different liquid-crystalline media should allow one to extract in a "model-free" way average orientations of dipolar vectors and specific aspects of their motions.     

Reaction mechanism in crystalline solids: kinetics and conformational dynamics of the Norrish type II biradicals from α-adamantyl-p-methoxyacetophenone

In an effort to determine the details of the solid-state reaction mechanism and diastereoselectivity in the Norrish type II and Yang cyclization of crystalline α-adamantyl-p-methoxyacetophenone, we determined its solid-state quantum yields and transient kinetics using nanocrystalline suspensions. The transient spectroscopy measurements were complemented with solid-state NMR spectroscopy spin-lattice relaxation experiments using isotopically labeled samples and with the analysis of variable-temperature anisotropic displacement parameters from single-crystal X-ray diffraction to determine the rate of interconversion of biradical conformers by rotation of the globular adamantyl group. Our experimental findings include a solid-state quantum yield for reaction that is 3 times greater than that in solution, a Norrish type II hydrogen-transfer reaction that is about 8 times faster in crystals than in solution, and a biradical decay that occurs on the same time scale as conformational exchange, which helps to explain the diastereoselectivity observed in the solid state.     

Revealing protein structures in solid-phase peptide synthesis by 13C solid-state NMR: evidence of excessive misfolding for Alzheimer's β

Solid-phase peptide synthesis (SPPS) is a widely used technique in biology and chemistry. However, the synthesis yield in SPPS often drops drastically for longer amino acid sequences, presumably because of the occurrence of incomplete coupling reactions. The underlying cause for this problem is hypothesized to be a sequence-dependent propensity to form secondary structures through protein aggregation. However, few methods are available to study the site-specific structure of proteins or long peptides that are anchored to the solid support used in SPPS. This study presents a novel solid-state NMR (SSNMR) approach to examine protein structure in the course of SPPS. As a useful benchmark, we describe the site-specific SSNMR structural characterization of the 40-residue Alzheimer's β-amyloid (Aβ) peptide during SPPS. Our 2D (13)C/(13)C correlation SSNMR data on Aβ(1-40) bound to a resin support demonstrated that Aβ underwent excessive misfolding into a highly ordered β-strand structure across the entire amino acid sequence during SPPS. This approach is likely to be applicable to a wide range of peptides/proteins bound to the solid support that are synthesized through SPPS.     

Variability of the 15N chemical shielding tensors in the B3 domain of protein G from 15N relaxation measurements at several fields. Implications for backbone order parameters

We applied a combination of 15N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 T) to determine the 15N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15N chemical shielding tensor, similar to those in ubiquitin (Fushman, et al. J. Am. Chem. Soc. 1998, 120, 10947; J. Am. Chem. Soc. 1999, 121, 8577). Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02 A NH bond length) and the site-to-site CSA variability is +/-17.6 to +/-21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H (Kroenke, et al. J. Am. Chem. Soc. 1999, 121, 10119) or recently, using "meta-analysis" for ubiquitin (Damberg, et al. J. Am. Chem. Soc. 2005, 127, 1995). Standard interpretation of 15N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15N CSA for accurate analysis of protein dynamics from 15N relaxation measurements.