ICMRBS founder’s medal 2006: Biological solid-state NMR,methods and applications

Solid-state NMR (ssNMR) provides increasing possibilities to study structure and dynamics of biomolecular systems. Our group has been interested in developing ssNMR-based approaches that are applicable to biomolecules of increasing molecular size and complexity without the need of specific isotope-labelling. Methodological aspects ranging from spectral assignments to the indirect detection of proton–proton contacts in multi-dimensional ssNMR are discussed and applied to (membrane) protein complexes.     

NMR methods for characterizing microsecond to millisecond dynamics in recognition and catalysis

During the past two years, significant advances have been made in the development of NMR methods for studying biomolecular dynamics on the microsecond to millisecond timescale. Applications of these methods to biologically relevant systems have provided compelling evidence that, in many cases, conformational dynamics on these timescales govern the rates of biomolecular recognition and catalysis.     

RFDR with Adiabatic Inversion Pulses: Application to Internuclear Distance Measurements

In the context of the structural characterisation of biomolecular systems via MAS solid state NMR, the potential utility of homonuclear dipolar recoupling with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. The results obtained suggest that it is possible to obtain reliable estimates of internuclear distances via an analysis of the initial cross-peak intensity buildup curves generated from two-dimensional adiabatic inversion pulse driven longitudinal magnetisation exchange experiments.     

NMR reveals novel mechanisms of protein activity regulation

NMR spectroscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide an integrated insight into both the structure and intrinsic dynamics of biomolecules. In addition, NMR can provide site-resolved information about the conformation entropy of binding, as well as about energetically excited conformational states. Recent advances have enabled the application of NMR for the characterization of supramolecular systems. A summary of mechanisms underpinning protein activity regulation revealed by the application of NMR spectroscopy in a number of biological systems studied in the lab is provided.     

Traceless and Site-specific Attachment of Proteins onto Solid Supports

Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.     

NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix–helix interactions in membrane proteins

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.     

Internal pH indicators for biomolecular NMR

We describe a non-invasive technique for determining pH in biomolecular NMR sample using buffer components (formate, tris, piperazine, and imidazole) as internal pH indicators, whose (1)H NMR chemical shifts are sensitive to pH in a range from 2.5 to 9.8. This method is suitable for a wide range of applications where samples are handled intensively during NMR titrations or in high throughput analysis in structural genomics or metabolomics.     

Solution NMR views of dynamical ordering of biomacromolecules

Background

To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells.

Novel NMR tools to study structure and dynamics of biomembranes

Nuclear magnetic resonance (NMR) studies on biomembranes have benefited greatly from introduction of magic angle spinning (MAS) NMR techniques. Improvements in MAS probe technology, combined with the higher magnetic field strength of modern instruments, enables almost liquid-like resolution of lipid resonances. The cross-relaxation rates measured by nuclear Overhauser enhancement spectroscopy (NOESY) provide new insights into conformation and dynamics of lipids with atomic-scale resolution. The data reflect the tremendous motional disorder in the lipid matrix. Transfer of magnetization by spin diffusion along the proton network of lipids is of secondary relevance, even at a long NOESY mixing time of 300 ms. MAS experiments with re-coupling of anisotropic interactions, like the 13C-(1)H dipolar couplings, benefit from the excellent resolution of 13C shifts that enables assignment of the couplings to specific carbon atoms. The traditional 2H NMR experiments on deuterated lipids have higher sensitivity when conducted on oriented samples at higher magnetic field strength. A very large number of NMR parameters from lipid bilayers is now accessible, providing information about conformation and dynamics for every lipid segment. The NMR methods have the sensitivity and resolution to study lipid-protein interaction, lateral lipid organization, and the location of solvents and drugs in the lipid matrix.     

Combined zero-quantum and spin-diffusion mixing for efficient homonuclear correlation spectroscopy under fast MAS: broadband recoupling and detection of long-range correlations

Fast magic angle spinning (MAS) NMR spectroscopy is emerging as an essential analytical and structural biology technique. Large resolution and sensitivity enhancements observed under fast MAS conditions enable structural and dynamics analysis of challenging systems, such as large macromolecular assemblies and isotopically dilute samples, using only a fraction of material required for conventional experiments. Homonuclear dipolar-based correlation spectroscopy constitutes a centerpiece in the MAS NMR methodological toolbox, and is used essentially in every biological and organic system for deriving resonance assignments and distance restraints information necessary for structural analysis. Under fast MAS conditions (rotation frequencies above 35–40 kHz), dipolar-based techniques that yield multi-bond correlations and non-trivial distance information are ineffective and suffer from low polarization transfer efficiency. To overcome this limitation, we have developed a family of experiments, CORD–RFDR. These experiments exploit the advantages of both zero-quantum RFDR and spin-diffusion based CORD methods, and exhibit highly efficient and broadband dipolar recoupling across the entire spectrum, for both short-range and long-range correlations. We have verified the performance of the CORD–RFDR sequences experimentally on a U-¹³C,¹⁵N-MLF tripeptide and by numerical simulations. We demonstrate applications of 2D CORD–RFDR correlation spectroscopy in dynein light chain LC8 and HIV-1 CA tubular assemblies. In the CORD–RFDR spectra of LC8 acquired at the MAS frequency of 40 kHz, many new intra- and inter-residue correlations are detected, which were not observed with conventional dipolar recoupling sequences. At a moderate MAS frequency of 14 kHz, the CORD–RFDR experiment exhibits excellent performance as well, as demonstrated in the HIV-1 CA tubular assemblies. Taken together, the results indicate that CORD–RFDR experiment is beneficial in a broad range of conditions, including both high and moderate MAS frequencies and magnetic fields.     

New Methodology for Computer-Aided Modelling of Biomolecular Structure and Dynamics 1. Non-Cyclic Structures

Abstract

A general methodology is proposed for the conformational modelling of biomolecular systems. The approach allows one: (i) to describe the system under investigation by an arbitrary set of internal variables, i.e., torsion angles, bond angles, and bond lengths; it offers a possibility to pass from the free structure to a completely fixed one with the number of variables from 3N to zero, respectively, where N is the number of atoms; (ii) to consider both, a single molecule and a complex of many molecules, (e.g., proteins, water, ligands, etc.) in terms of one universal model; (iii) to study the dynamics of the system using explicit analytical Lagrangian equations of motion, thus opening up possibilities for investigations of slow concerted motions such as domain oscillations in proteins etc.; (iv) to calculate the partial derivatives of various functions of conformation, e.g., the conformatinal energy or external constraints imposed, using a standard efficient procedure regardless of the variables and the structure of the system. The approach is meant to be used in various investigations concerning the conformations and dynamics of biomacromolecules.     

High performance computing in biology: multimillion atom simulations of nanoscale systems

Computational methods have been used in biology for sequence analysis (bioinformatics), all-atom simulation (molecular dynamics and quantum calculations), and more recently for modeling biological networks (systems biology). Of these three techniques, all-atom simulation is currently the most computationally demanding, in terms of compute load, communication speed, and memory load. Breakthroughs in electrostatic force calculation and dynamic load balancing have enabled molecular dynamics simulations of large biomolecular complexes. Here, we report simulation results for the ribosome, using approximately 2.64 million atoms, the largest all-atom biomolecular simulation published to date. Several other nano-scale systems with different numbers of atoms were studied to measure the performance of the NAMD molecular dynamics simulation program on the Los Alamos National Laboratory Q Machine. We demonstrate that multimillion atom systems represent a 'sweet spot' for the NAMD code on large supercomputers. NAMD displays an unprecedented 85% parallel scaling efficiency for the ribosome system on 1024 CPUs. We also review recent targeted molecular dynamics simulations of the ribosome that prove useful for studying conformational changes of this large biomolecular complex in atomic detail.     

Increasing the Time Resolution of Single-Molecule Experiments with Bayesian Inference

Many time-resolved single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here, we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian inference for the analysis of subtemporal resolution dynamics approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data.     

Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state (31)P and (13)C NMR spectroscopy

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.     

核磁共振波谱应用于结构生物学的研究进展

综述了核磁共振波谱在结构生物学研究中的进展。在溶液中测定生物大分子的结构,分子大小的限制正被减少,尽管新结构的测定仍然需要付出比较大的努力。核磁共振是一个有效的手段,可用于研究在许多细胞过程中存在的弱的或者瞬态的蛋白质-蛋白质相互作用。结构的柔性在蛋白质分子功能中起了中心作用。由于最近方法学的发展,使NMR可以表征蛋白质的动力学,从而可以对分子机制有新的认识。核磁共振波谱可以在原子分辨率下表征无序的蛋白质系统,可以研究折叠路径。跨膜蛋白在细胞中起了关键作用,这使它们成为药物的靶标。应用液体和固体核磁共振技术已经成功测定了跨膜蛋白质的结构。     

Residual dipolar couplings: Synergy between NMR and structural genomics

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.     

High-Resolution 1H NMR Spectroscopy of Fish Muscle,Eggs and Small Whole Fish via Hadamard-Encoded Intermolecular Multiple-Quantum Coherence

Background and Purpose

Nuclear magnetic resonance (NMR) spectroscopy has become an important technique for tissue studies. Since tissues are in semisolid-state, their high-resolution (HR) spectra cannot be obtained by conventional NMR spectroscopy. Because of this restriction, extraction and high-resolution magic angle spinning (HR MAS) are widely applied for HR NMR spectra of tissues. However, both of the methods are subject to limitations. In this study, the feasibility of HR ¹H NMR spectroscopy based on intermolecular multiple-quantum coherence (iMQC) technique is explored using fish muscle, fish eggs, and a whole fish as examples.

Materials and Methods

Intact salmon muscle tissues, intact eggs from shishamo smelt and a whole fish (Siamese algae eater) are studied by using conventional 1D one-pulse sequence, Hadamard-encoded iMQC sequence, and HR MAS.

Results

When we use the conventional 1D one-pulse sequence, hardly any useful spectral information can be obtained due to the severe field inhomogeneity. By contrast, HR NMR spectra can be obtained in a short period of time by using the Hadamard-encoded iMQC method without shimming. Most signals from fatty acids and small metabolites can be observed. Compared to HR MAS, the iMQC method is non-invasive, but the resolution and the sensitivity of resulting spectra are not as high as those of HR MAS spectra.

Conclusion

Due to the immunity to field inhomogeneity, the iMQC technique can be a proper supplement to HR MAS, and it provides an alternative for the investigation in cases with field distortions and with samples unsuitable for spinning. The acquisition time of the proposed method is greatly reduced by introduction of the Hadamard-encoded technique, in comparison with that of conventional iMQC method.