TROSY in NMR studies of the structure and function of large biological macromolecules

Transverse relaxation-optimized spectroscopy (TROSY), in combination with various isotope-labeling techniques, has opened avenues to study biomolecules with molecular masses of up to 1000000Da by solution NMR. Important recent applications of TROSY include the structure determination of membrane proteins in detergent micelles, structural and functional studies of large proteins in both monomeric form and macromolecular complexes, and investigations of intermolecular interactions in large complexes. TROSY improves the measurement of residual dipolar couplings and the detection of scalar couplings across hydrogen bonds - techniques that promise to further enhance the determination of solution structures of large proteins and oligonucleotides.     

X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution

Crystallography supplies unparalleled detail on structural information critical for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. Small angle X-ray scattering (SAXS) offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 A to 10 A resolution, but without the size limitations inherent in NMR and electron microscopy studies. Together these techniques can allow multi-scale modeling to create complete and accurate images of macromolecules for modeling allosteric mechanisms, supramolecular complexes, and dynamic molecular machines acting in diverse processes ranging from eukaryotic DNA replication, recombination and repair to microbial membrane secretion and assembly systems. This review addresses both theoretical and practical concepts, concerns and considerations for using these techniques in conjunction with computational methods to productively combine solution scattering data with high-resolution structures. Detailed aspects of SAXS experimental results are considered with a focus on data interpretation tools suitable to model protein and nucleic acid macromolecular structures, including membrane protein, RNA, DNA, and protein-nucleic acid complexes. The methods discussed provide the basis to examine molecular interactions in solution and to study macromolecular flexibility and conformational changes that have become increasingly relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.     

Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.     

Solution NMR studies of the integral membrane proteins OmpX and OmpA from Escherichia coli

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA beta-barrel from those of OmpX. Overall, the present data demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.     

NMR analysis of methyl groups at 100-500 kDa: model systems and Arp2/3 complex

Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses. Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range. Biochemical methods also must be developed in concert to produce such systems in selectively labeled form. Here, we present (1)H/(13)C-HSQC spectra of protonated methyl groups in a model system that mimics molecular weights up to approximately 560 kDa. Signals from side chain methyl groups of Ile, Leu, and Val residues are clearly detectable at correlation times up to approximately 330 ns. We have also developed a biochemical procedure to produce the 240 kDa, heteroheptameric Arp2/3 actin nucleation complex selectively labeled at one subunit and obtained (1)H/(13)C-HSQC spectra of this assembly. Sensitivity in spectra of both the Arp2/3 complex and the model system indicate that methyl groups will be useful sources of information in nonsymmetric systems with molecular weights greater than 600 kDa at concentrations less than 100 microM. Methyl analyses will complement TROSY and CRINEPT analyses of amides in NMR studies of structure and molecular interactions of extremely large macromolecules and assemblies.     

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote <Emphasis Type="Italic">Pichia pastoris</Emphasis>

¹³C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific ¹³C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient ¹³C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.     

Analytical ultracentrifugation combined with X-ray and neutron scattering: Experiment and modelling

Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure.     

Geometry dependent two-dimensional heteronuclear multiplet effects in paramagnetic proteins

We report experimental observation and numerical simulation of a two-dimensional multiplet effect in the heteronuclear correlation spectrum of a paramagnetic protein that depends on molecular geometry. This effect arises as a consequence of cross-correlated relaxation involving the Curie spin relaxation and internuclear dipolar relaxation mechanisms. It also manifests itself in resolution and sensitivity improvement in transverse relaxation optimised spectroscopy (TROSY) kind of experiments. Characteristic multiplet patterns in heteronuclear coupled two-dimensional NMR spectra encode directional information for the heteronuclear bond with respect to the paramagnetic center. These patterns, which are simulated here using Redfield's relaxation theory, can be used to obtain a new type of geometry restriction for structure determination and refinement of paramagnetic macromolecular systems.     

NMR spectroscopy to study the fate of metallodrugs in cells

Metal-based drugs can modulate various biological processes and exhibit a rich variety of properties that foster their use in biomedicine and chemical biology. On the way to intracellular targets, ligand exchange and redox reactions can take place, thus making metallodrug speciation in vivo a challenging task. Advances in NMR spectroscopy have made it possible to move from solution to live-cell studies and elucidate the transport of metallodrugs and interactions with macromolecular targets in a physiological setting. In turn, the electronic properties and supramolecular chemistry of metal complexes can be exploited to characterize drug delivery nanosystems by NMR. The recent evolution of in-cell NMR methodology is presented with special emphasis on metal-related processes. Applications to paradigmatic cases of platinum and gold drugs are highlighted.     

A novel NMR method for determining the interfaces of large protein-protein complexes

Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.     

An Isotope Labeling Strategy for Methyl TROSY Spectroscopy

Recently we have shown that HMQC spectra of protonated methyl groups in high molecular weight, highly deuterated proteins have large enhancements in sensitivity and resolution relative to HSQC-generated data sets. These enhancements derive from a TROSY effect in which complete cancellation of intra-methyl (1)H-(1)H and (1)H-(13)C dipolar interactions occurs for 50% of the signal in the case of HMQC, so long as the methyl is attached to a molecule tumbling in the macromolecular limit (Tugarinov, V., Hwang, P.M., Ollerenshaw, J.E., Kay, L.E. J. Am. Chem. Soc. (2003) 125, 10420-10428; Ollerenshaw, J.E., Tugarinov, V. and Kay, L.E. Magn. Reson. Chem. (2003) 41, 843-852. The first demonstration of this effect was made for isoleucine delta1 methyl groups in a highly deuterated 82 kDa protein, malate synthase G. As with (1)H-(15)N TROSY spectroscopy high levels of deuteration are critical for maximizing the TROSY effect. Here we show that excellent quality methyl TROSY spectra can be recorded on U-[(2)H] Iledelta1-[(13)CH(3)] Leu,Val-[(13)CH(3)/(12)CD(3)] protein samples, significantly extending the number of probes available for structural and dynamic studies of high molecular weight systems.     

NMR studies of conformational states and dynamics of DNA

The application of high resolution NMR techniques to the investigation of DNA double helices in solution is currently in a rapid state of change as a result of advances in three different fields. First, new methods (cloning, enzymatic degradation, sonication, and chemical synthesis) have been developed for producing large quantities of short DNA suitable for NMR studies. Second, there have been major advances in the field of NMR in terms of the introduction of new pulse techniques and improvements in instrumentation. Finally, as a result of recent X-ray diffraction studies on short DNA helices and the discovery of left-handed Z-DNA there is heightened interest in the study of DNA structures in solution and the effect of sequence on structure. In the present review, we discuss the way in which NMR techniques have been used to probe various aspects of the DNA properties, including base pairing structure, dynamics of breathing, effect of sequence on DNA structure, internal molecular motions, the effect of environment on the DNA, and the interaction of DNA with small ligands.     

The future is hybrid

On the occasion of the 50th anniversary of the Journal of Structural Biology, we review some of the major advances that have taken place in molecular and cellular structural biology over this timeframe and consider some current trends, as well as promising new directions. While the primary experimental techniques of X-ray diffraction, electron microscopy and NMR spectroscopy continue to improve and other powerful new techniques have come on-line, it appears that the most comprehensive analyses of large, dynamic, macromolecular machines will rely on integrated combinations of different methodologies, viz. "hybrid approaches". The same prospect applies to the challenge of integrating observations of isolated macromolecules with data pertaining to their distributions and interaction networks in living cells. Looking ahead, computation in its diverse aspects may be expected to assume an increasingly important role in structural biology, as the prediction of molecular structures, the computation of dynamic properties, and quantitative time-resolved models of intracellular molecular populations (structural systems biology) move towards functional maturity.     

蛋白质溶液NMR结构测定的一些新进展

新的标记技术的进展和采用稀释的液晶作为溶剂以提供额外的结构信息,提高了核磁共振技术测定蛋白质溶液三维结构的精度,扩大了分子质量测定范围.目前已经利用多维 ¹⁵N,¹³C,²H标记NMR测定了许多分子质量为30 ku左右的蛋白质溶液结构,这一上限可能还会被进一步提高.     

A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data

We present a novel and efficient approach for assessing protein-protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed "restrained soft-docking," is validated for several known protein complexes. These data demonstrate that restrained soft-docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.     

Recent excitements in protein NMR: Large proteins and biologically relevant dynamics

The advent of Transverse Relaxation Optimized SpectroscopY (TROSY) and perdeuteration allowed biomolecular NMR spectroscopists to overcome the size limitation barrier (~20 kDa) in de novo structure determination of proteins. The utility of these techniques was immediately demonstrated on large proteins and protein complexes (e.g. GroEL-GroES, ClpP protease, Hsp90-p53, 20S proteasome, etc.). Further, recent methodological developments such as Residual Dipolar Couplings and Paramagnetic Relaxation Enhancement allowed accurate measurement of long-range structural restraints. Additionally, Carr-Purcell-Meiboom-Gill (CPMG), rotating frame relaxation experiments (R₁ρ) and saturation transfer experiments (CEST and DEST) created never-before accessibility to the μs–ms timescale dynamic parameters that led to the deeper understanding of biological processes. Meanwhile, the excitement in the field continued with a series of developments in the fast data acquisition methods allowing rapid structural studies on less stable proteins. This review aims to discuss important developments in the field of biomolecular NMR spectroscopy in the recent past, i.e., in the post TROSY era. These developments not only gave access to the structural studies of large protein assemblies, but also revolutionized tools in the arsenal of today’s biomolecular NMR and point to a bright future of biomolecular NMR spectroscopy.     

Differences in Cellulosic Supramolecular Structure of Compositionally Similar Rice Straw Affect Biomass Metabolism by Paddy Soil Microbiota

Because they are strong and stable, lignocellulosic supramolecular structures in plant cell walls are resistant to decomposition. However, they can be degraded and recycled by soil microbiota. Little is known about the biomass degradation profiles of complex microbiota based on differences in cellulosic supramolecular structures without compositional variations. Here, we characterized and evaluated the cellulosic supramolecular structures and composition of rice straw biomass processed under different milling conditions. We used a range of techniques including solid- and solution-state nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy followed by thermodynamic and microbial degradability characterization using thermogravimetric analysis, solution-state NMR, and denaturing gradient gel electrophoresis. These measured data were further analyzed using an “ECOMICS” web-based toolkit. From the results, we found that physical pretreatment of rice straw alters the lignocellulosic supramolecular structure by cleaving significant molecular lignocellulose bonds. The transformation from crystalline to amorphous cellulose shifted the thermal degradation profiles to lower temperatures. In addition, pretreated rice straw samples developed different microbiota profiles with different metabolic dynamics during the biomass degradation process. This is the first report to comprehensively characterize the structure, composition, and thermal degradation and microbiota profiles using the ECOMICS toolkit. By revealing differences between lignocellulosic supramolecular structures of biomass processed under different milling conditions, our analysis revealed how the characteristic compositions of microbiota profiles develop in addition to their metabolic profiles and dynamics during biomass degradation.     

The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments

Solid-state NMR spectroscopy is a powerful technique for the investigation of membrane-associated peptides and proteins as well as their interactions with lipids, and a variety of conceptually different approaches have been developed for their study. The technique is unique in allowing for the high-resolution investigation of liquid disordered lipid bilayers representing well the characteristics of natural membranes. Whereas magic angle solid-state NMR spectroscopy follows approaches that are related to those developed for solution NMR spectroscopy the use of static uniaxially oriented samples results in angular constraints which also provide information for the detailed analysis of polypeptide structures. This review introduces this latter concept theoretically and provides a number of examples. Furthermore, ongoing developments combining solid-state NMR spectroscopy with information from solution NMR spectroscopy and molecular modelling as well as exploratory studies using dynamic nuclear polarization solid-state NMR will be presented.