RNA Dynamics by NMR Spectroscopy

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined—Lego like—than in proteins, hence the numerous revelations of RNA excited states.     

The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

¹⁵N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H^N–H^α coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (λ lysozyme) in solution. The lower and upper lip regions in λ lysozyme (residues 51–60 and 128–141, respectively) show reduced ¹H–¹⁵N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T₂ indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for λ lysozyme indicate that two fluctuating β‐strands (β3 and β4) are populated in the lower lip region while the N terminus of helix α6 (residues 136–139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the β‐sheet and helical secondary structure in the lip regions, with populations of 40–60 %. Thus in solution λ lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein.     

Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non-hydrolyzable analogues GppNHp and GppCH₂p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors.     

A Method for Systematic Assessment of Intrinsically Disordered Protein Regions by NMR

Intrinsically disordered proteins (IDPs) that lack stable conformations and are highly flexible have attracted the attention of biologists. Therefore, the development of a systematic method to identify polypeptide regions that are unstructured in solution is important. We have designed an “indirect/reflected” detection system for evaluating the physicochemical properties of IDPs using nuclear magnetic resonance (NMR). This approach employs a “chimeric membrane protein”-based method using the thermostable membrane protein PH0471. This protein contains two domains, a transmembrane helical region and a C-terminal OB (oligonucleotide/oligosaccharide binding)-fold domain (named NfeDC domain), connected by a flexible linker. NMR signals of the OB-fold domain of detergent-solubilized PH0471 are observed because of the flexibility of the linker region. In this study, the linker region was substituted with target IDPs. Fifty-three candidates were selected using the prediction tool POODLE and 35 expression vectors were constructed. Subsequently, we obtained ¹⁵N-labeled chimeric PH0471 proteins with 25 IDPs as linkers. The NMR spectra allowed us to classify IDPs into three categories: flexible, moderately flexible, and inflexible. The inflexible IDPs contain membrane-associating or aggregation-prone sequences. This is the first attempt to use an indirect/reflected NMR method to evaluate IDPs and can verify the predictions derived from our computational tools.     

Normal Mode Dynamics Comparison of Proteins

Sequence and structure comparisons are fundamental techniques that enable exploration of the sequence and structural spaces of proteins. Homology detection, function prediction, and protein classification rely on these techniques. However, protein structures are dynamic, rather than static, and such protein dynamics play a key role in a wide range of biological activities. Therefore, protein dynamics comparison algorithms may shed light on the relationship between proteins′ dynamics and function. Here, we review different strategies for comparing dynamics of proteins. Special emphasis is given to newly developed algorithms that compare dynamics of proteins with no apparent sequence or structural similarity and to the qualitative differences between these algorithms.     

Biomolecular Structure and Dynamics with Neutrons: The View from Simulation

The characterization of the structure and internal dynamics of biomolecules is essential to understanding their biological function. Neutron scattering probes similar time- and length-scales to molecular dynamics simulation. Hence, simulation models of biomolecules have become invaluable in the interpretation of experimental neutron data. Here, we report on advances in the application of simulation in developing neutron scattering to investigate internal protein motions and, as an example of industrial relevance, in the derivation of physical models of use in biofuel renewable energy research.     

A Review on Combination of Ab Initio Molecular Dynamics and NMR Parameters Calculations

This review focuses on a combination of ab initio molecular dynamics (aiMD) and NMR parameters calculations using quantum mechanical methods. The advantages of such an approach in comparison to the commonly applied computations for the structures optimized at 0 K are presented. This article was designed as a convenient overview of the applied parameters such as the aiMD type, DFT functional, time step, or total simulation time, as well as examples of previously studied systems. From the analysis of the published works describing the applications of such combinations, it was concluded that including fast, small-amplitude motions through aiMD has a noticeable effect on the accuracy of NMR parameters calculations.     

Solution Structure and Dynamics of the Small Protein HVO_2922 from Haloferax volcanii

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation.     

Relaxation Dynamics and Strain Persistency of Azobenzene‐Functionalized Polymers and Actuators

The accumulation of photoinduced deformation in azobenzene functionalized polymers has received a significant amount of attention in recent years. Critically, the induced photomechanical deformation in these systems experiences varying degrees of relaxation. Control over the persistence of photomechanical strains is vital to the broader utility of these materials in shape programmable systems including soft robotics and engineered origami. Furthermore, investigations of relaxation in light responsive polymer systems triggered by UV light are more prominent than those triggered by blue‐green light. In this study, the impact of chain mobility and initially induced photostrain on the relaxation dynamics of azobenzene‐functionalized polyimides after irradiation with blue light is examined. A modeling effort coupling chromophore population dynamics to material strain is carried out to further explore the relationship between material structure, relaxation dynamics, and macroscopic deformation. The implications for controlling strain persistence are highlighted by simulating one example of a photoprimed bistable actuator.     

Water Dynamics in Whey-Protein-Based Composite Hydrogels by Means of NMR Relaxometry

Whey-protein-isolate-based composite hydrogels with encapsulated black carrot (Daucus carota) extract were prepared by heat-induced gelation. The hydrogels were blended with gum tragacanth, pectin and xanthan gum polysaccharides for modulating their properties. ¹H spin-lattice relaxation experiments were performed in a broad frequency range, from 4 kHz to 30 MHz, to obtain insight into the influence of the different polysaccharides and of the presence of black carrot on dynamical properties of water molecules in the hydrogel network. The ¹H spin-lattice relaxation data were decomposed into relaxation contributions associated with confined and free water fractions. The population of the confined water fraction and the value of the translation diffusion coefficient of water molecules in the vicinity of the macromolecular network were quantitatively determined on the basis of the relaxation data. Moreover, it was demonstrated that the translation diffusion is highly anisotropic (two-dimensional, 2D).     

Probing molecular mobility during crosslinking process of commercial resins by NMR multiexponential relaxation data analysis

In this study, we present the experimental results for the crosslinking process of a commercial polyester resin based on measurements of the spin lattice relaxation time T₁ of protons, as function of the crosslinking time evolution. Multiexponential decomposition of the evolution of magnetization measured in inversion‐recovery experiments is performed. The population of “rigid” and “mobile” nuclear spin sites was estimated as function of time evolution. In analogy to the usual monomer conversion u, site conversion from “mobile” to “rigid” sites u_M were also estimated as a function of time evolution and initial concentrations of the reagents. The multiexponential decomposition approach of T₁ relaxation data allows one to follow crosslinking processes. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore’s inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.     

Nanobody Paratope Ensembles in Solution Characterized by MD Simulations and NMR

Variable domains of camelid antibodies (so-called nanobodies or V_HH) are the smallest antibody fragments that retain complete functionality and therapeutic potential. Understanding of the nanobody-binding interface has become a pre-requisite for rational antibody design and engineering. The nanobody-binding interface consists of up to three hypervariable loops, known as the CDR loops. Here, we structurally and dynamically characterize the conformational diversity of an anti-GFP-binding nanobody by using molecular dynamics simulations in combination with experimentally derived data from nuclear magnetic resonance (NMR) spectroscopy. The NMR data contain both structural and dynamic information resolved at various timescales, which allows an assessment of the quality of protein MD simulations. Thus, in this study, we compared the ensembles for the anti-GFP-binding nanobody obtained from MD simulations with results from NMR. We find excellent agreement of the NOE-derived distance maps obtained from NMR and MD simulations and observe similar conformational spaces for the simulations with and without NOE time-averaged restraints. We also compare the measured and calculated order parameters and find generally good agreement for the motions observed in the ps–ns timescale, in particular for the CDR3 loop. Understanding of the CDR3 loop dynamics is especially critical for nanobodies, as this loop is typically critical for antigen recognition.     

NMR Assessment of Therapeutic Peptides and Proteins: Correlations That Reveal Interactions and Motions

NMR measurements of rotational and translational diffusion are used to characterize the solution behavior of a wide variety of therapeutic proteins and peptides. The timescales of motions sampled in these experiments reveal complicated intrinsic solution behavior such as flexibility, that is central to function, as well as self-interactions, stress-induced conformational changes and other critical attributes that can be discovery and development liabilities. Trends from proton transverse relaxation (R₂) and hydrodynamic radius (R_h) are correlated and used to identify and differentiate intermolecular from intramolecular interactions. In this study, peptide behavior is consistent with complicated multimer self-assembly, while multi-domain protein behavior is dominated by intramolecular interactions. These observations are supplemented by simulations that include effects from slow transient interactions and rapid internal motions. R₂–R_h correlations provide a means to profile protein motions as well as interactions. The approach is completely general and can be applied to therapeutic and target protein characterization.     

NMR properties of poly(dimethyl siloxane) colloids as new contrast agents for NMR imaging

By connecting the field‐gradient spin‐echo theory to spin–spin relaxation, we have found that the relationship between the tube‐reptation model and spin–spin relaxation can be represented by G(t) = exp[−(t/T₂) ⁿ] in which n = 1 and 0.5 for regimes IV and III, respectively. In our experiments, the spin–spin relaxation of linear poly(dimethyl siloxane) (PDMS) agrees with G(t) = exp[−(t/T₂)] while that of crosslinked PDMS coincides with G(t) = exp[−(t/T₂)^0.5]. These results reflect that in the time interval 8–800 ms the dynamics of linear PDMS are in regime IV (governed by reptation motions) and those of the crosslinked PDMS are in regime III (dominated by wriggling motions). The line‐shapes of NMR spectra of crosslinked PDMS are consistent with the Lorentzian rather than the Gaussian model. This can be accounted for by supposing that the PDMS chains between crosslinks have liquid‐like motions even though crosslinked PDMS is a solid. The liquid‐like motions of crosslinked PDMS could be regarded as wriggling motions described by the tube‐reptation model. In addition, the experimental results of diameter distribution, viscosity, NMR image and spin–lattice relaxation are presented in this work. © 2000 Society of Chemical Industry     

Local Dynamics of Bismaleimide Adhesives in an Aggressive Environment

Molecular aspects of chemical and physical changes in bismaleimide (BMI) adhesive joints caused by absorbed moisture were investigated. The focus was on the early (pre-damage) stage that precedes the formation of voids and microcracks. Local dynamics were investigated by broad-band dielectric relaxation spectroscopy (DRS) and the changes in the chemical state of the matter were monitored by Fourier transform infrared spectroscopy (FTIR). Absorbed water interacts with the BMI network and gives rise to a fast relaxation process (termed γ*), characterized by an increase in the dielectric relaxation strength, an Arrhenius temperature dependence of the average relaxation time, and an activation energy of 50 kJ/mol. The γ* dynamics are slower than the relaxation of bulk liquid water because of the interactions between the absorbed water and various sites on the network (the ether oxygen, the hydroxyl group, the carbonyl group, and the tertiary amine nitrogen). One particularly significant finding is that the average relaxation time for the γ* process above 20°C is of the order of nanoseconds or less and, hence, the detection and monitoring of this process hinges upon the ability to perform high precision DRS at frequencies above 1 MHz. This is an important consideration in the ongoing efforts aimed at the implementation of DRS as a non-destructive inspection (NDI) tool for adhesive joints. FTIR spectra reveal the presence of non hydrogen-bonded water and hydrogen-bonded water, the latter bonded to one and/or two sites on the BMI network. A good agreement was found between the calculated ratio of non hydrogen-bonded to total absorbed water from DRS and FTIR data.     

Local Dynamics of Bismaleimide Adhesives in an Aggressive Environment

Molecular aspects of chemical and physical changes in bismaleimide (BMI) adhesive joints caused by absorbed moisture were investigated. The focus was on the early (pre-damage) stage that precedes the formation of voids and microcracks. Local dynamics were investigated by broad-band dielectric relaxation spectroscopy (DRS) and the changes in the chemical state of the matter were monitored by Fourier transform infrared spectroscopy (FTIR). Absorbed water interacts with the BMI network and gives rise to a fast relaxation process (termed γ*), characterized by an increase in the dielectric relaxation strength, an Arrhenius temperature dependence of the average relaxation time, and an activation energy of 50 kJ/mol. The γ* dynamics are slower than the relaxation of bulk liquid water because of the interactions between the absorbed water and various sites on the network (the ether oxygen, the hydroxyl group, the carbonyl group, and the tertiary amine nitrogen). One particularly significant finding is that the average relaxation time for the γ* process above 20°C is of the order of nanoseconds or less and, hence, the detection and monitoring of this process hinges upon the ability to perform high precision DRS at frequencies above 1 MHz. This is an important consideration in the ongoing efforts aimed at the implementation of DRS as a non-destructive inspection (NDI) tool for adhesive joints. FTIR spectra reveal the presence of non hydrogen-bonded water and hydrogen-bonded water, the latter bonded to one and/or two sites on the BMI network. A good agreement was found between the calculated ratio of non hydrogen-bonded to total absorbed water from DRS and FTIR data.     

Ligand- and Structure-Based Approaches of Escherichia coli FabI Inhibition by Triclosan Derivatives: From Chemical Similarity to Protein Dynamics Influence

Enoyl-acyl carrier protein reductase (FabI) is the limiting step to complete the elongation cycle in type II fatty acid synthase (FAS) systems and is a relevant target for antibacterial drugs. E. coli FabI has been employed as a model to develop new inhibitors against FAS, especially triclosan and diphenyl ether derivatives. Chemical similarity models (CSM) were used to understand which features were relevant for FabI inhibition. Exhaustive screening of different CSM parameter combinations featured chemical groups, such as the hydroxy group, as relevant to distinguish between active/decoy compounds. Those chemical features can interact with the catalytic Tyr156. Further molecular dynamics simulation of FabI revealed the ionization state as a relevant for ligand stability. Also, our models point the balance between potency and the occupancy of the hydrophobic pocket. This work discusses the strengths and weak points of each technique, highlighting the importance of complementarity among approaches to elucidate EcFabI inhibitor's binding mode and offers insights for future drug discovery.     

Carbohydrate-protein interactions: a 3D view by NMR

This review focuses on the application of NMR methods for understanding, at the molecular and atomic levels, the diverse mechanisms by which sugar molecules are recognised by the binding sites of lectins, antibodies and enzymes. Given the intrinsic chemical natures of sugars and their flexibility, it is well established that NMR parameters should be complemented by computational methods in attempts to unravel the structural and conformational features of the molecular recognition process unambiguously. We therefore aim here to describe new and significant advances in the knowledge of carbohydrate-protein interactions, obtained by employing state-of-the-art NMR and molecular modelling. We have not attempted to prepare an exhaustive review but have tried to focus on describing the key aspects that should be considered when tackling a problem within this research topic.