Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.     

Prediction of Protein Structure Using Surface Accessibility Data

An approach to the de novo structure prediction of proteins is described that relies on surface accessibility data from NMR paramagnetic relaxation enhancements by a soluble paramagnetic compound (sPRE). This method exploits the distance‐to‐surface information encoded in the sPRE data in the chemical shift‐based CS‐Rosetta de novo structure prediction framework to generate reliable structural models. For several proteins, it is demonstrated that surface accessibility data is an excellent measure of the correct protein fold in the early stages of the computational folding algorithm and significantly improves accuracy and convergence of the standard Rosetta structure prediction approach.     

Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag

Site‐specific labeling of proteins with lanthanide ions offers great opportunities for investigating the structure, function, and dynamics of proteins by virtue of the unique properties of lanthanides. Lanthanide‐tagged proteins can be studied by NMR, X‐ray, fluorescence, and EPR spectroscopy. However, the rigidity of a lanthanide tag in labeling of proteins plays a key role in the determination of protein structures and interactions. Pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) are valuable long‐range structure restraints in structural‐biology NMR spectroscopy. Generation of these paramagnetic restraints generally relies on site‐specific tagging of the target proteins with paramagnetic species. To avoid nonspecific interaction between the target protein and paramagnetic tag and achieve reliable paramagnetic effects, the rigidity, stability, and size of lanthanide tag is highly important in paramagnetic labeling of proteins. Here 4′‐mercapto‐2,2′: 6′,2′′‐terpyridine‐6,6′′‐dicarboxylic acid (4MTDA) is introduced as a a rigid paramagnetic and fluorescent tag which can be site‐specifically attached to a protein by formation of a disulfide bond. 4MTDA can be readily immobilized by coordination of the protein side chain to the lanthanide ion. Large PCSs and RDCs were observed for 4MTDA‐tagged proteins in complexes with paramagnetic lanthanide ions. At an excitation wavelength of 340 nm, the complex formed by protein–4MTDA and Tb³⁺ produces high fluorescence with the main emission at 545 nm. These interesting features of 4MTDA make it a very promising tag that can be exploited in NMR, fluorescence, and EPR spectroscopic studies on protein structure, interaction, and dynamics.     

Protein Structural Ensembles Visualized by Solvent Paramagnetic Relaxation Enhancement

A protein can be in different conformations when fulfilling its function. Yet depiction of protein structural ensembles remains difficult. Here we show that the accurate measurement of solvent paramagnetic relaxation enhancement (sPRE) in the presence of an inert paramagnetic cosolute allows the assessment of protein dynamics. Demonstrated with two multi‐domain proteins, we present a method to characterize protein microsecond–millisecond dynamics based on the analysis of the sPRE. Provided with the known structures of a protein, our method uncovers an ensemble of structures that fully accounts for the observed sPRE. In conjunction with molecular dynamics simulations, our method can identify protein alternative conformation that has only been theorized before. Together, our method expands the application of sPRE beyond structural characterization of rigid proteins and complements the established PRE NMR technique.     

Backbone-only protein solution structures with a combination of classical and paramagnetism-based constraints: a method that can be scaled to large molecules.

Herein, it is shown that a medium-resolution solution structure of a protein can be obtained with the sole assignment of the protein backbone and backbone-related constriants if a derivative with a firmly bound paramagnetic metal is available. The proof-of-concept is provided on calbindin D9k, a calcium binding protein in which one of the two calcium ions can be selectively substituted by a paramagnetic lanthanide ion. The constraints used are HN (and Ha) nuclear Overhauser effects (NOEs), hydrogen bonds, dihedral angle constriants from chemical shifts, and the following paramagnetism-based constraints: 1) pseudocontact shifts, acquired by substituting one (or more) lanthanide(s) in the C-terminal calcium binding site; 2) N-HN residual dipolar couplings due to self-orientation induced by the paramagnetic lanthanide(s); 3) cross-correlations between the Curie and internuclear dipole-dipole interactions; and 4) paramagnetism-induced relaxation rate enhancements. An upper distance limit for internuclear distances between any two backbone atoms was also given according to the molecular weight of the protein. For this purpose, the paramagnetism-based constraints were collectively implemented in the program CYANA for solution structure determinations, similarly to what was previously done for the program DYANA. The method is intrinsically suitable for large molecular weight proteins.     

Structure determination of protein-ligand complexes by transferred paramagnetic shifts

Rational drug design depends on the knowledge of the three-dimensional (3D) structure of complexes between proteins and lead compounds of low molecular weight. A novel nuclear magnetic resonance (NMR) spectroscopy strategy based on the paramagnetic effects from lanthanide ions allows the rapid determination of the 3D structure of a small ligand molecule bound to its protein target in solution and, simultaneously, its location and orientation with respect to the protein. The method relies on the presence of a lanthanide ion in the protein target and on fast exchange between bound and free ligand. The binding affinity of the ligand and the paramagnetic effects experienced in the bound state are derived from concentration-dependent (1)H and (13)C spectra of the ligand at natural isotopic abundance. Combined with prior knowledge of the crystal or solution structure of the protein and of the magnetic susceptibility tensor of the lanthanide ion, the paramagnetic data define the location and orientation of the bound ligand molecule with respect to the protein from simple 1D NMR spectra. The method was verified with the ternary 30 kDa complex between the lanthanide-labeled N-terminal domain of the epsilon exonuclease subunit from the Escherichia coli DNA polymerase III, the subunit theta, and thymidine. The binding mode of thymidine was found to be very similar to that of thymidine monophosphate present in the crystal structure.     

Visualizing an Ultra‐Weak Protein–Protein Interaction in Phosphorylation Signaling

Proteins interact with each other to fulfill their functions. The importance of weak protein–protein interactions has been increasingly recognized. However, owing to technical difficulties, ultra‐weak interactions remain to be characterized. Phosphorylation can take place via a K_D≈25 mM interaction between two bacterial enzymes. Using paramagnetic NMR spectroscopy and with the introduction of a novel Gd^III‐based probe, we determined the structure of the resulting complex to atomic resolution. The structure accounts for the mechanism of phosphoryl transfer between the two enzymes and demonstrates the physical basis for their ultra‐weak interaction. Further, molecular dynamics (MD) simulations suggest that the complex has a lifetime in the micro‐ to millisecond regimen. Hence such interaction is termed a fleeting interaction. From mathematical modeling, we propose that an ultra‐weak fleeting interaction enables rapid flux of phosphoryl signal, providing a high effective protein concentration.     

Construction of a Metal‐Free Electron Spin System by Encapsulation of an NO Molecule Inside an Open‐Cage Fullerene C60 Derivative

A reactive radical species, nitric oxide (NO), was encapsulated in a unimolecular form inside an open‐cage fullerene derivative under high‐pressure conditions in the solid state. Surprisingly, the molecular complex showed sharp ¹H NMR signals despite the existence of the paramagnetic species inside the carbon cage. Owing to the paramagnetic shifts, the escape rate of the NO was determined experimentally. After constructing a stopper on the rim of the opening, the NO was found to stay inside the cage even at 50 °C. The ESR measurements of the powdery sample showed paramagnetic properties at low temperature. The single‐crystal X‐ray structure analysis clearly demonstrated the existence of the encapsulated NO molecule, suggesting rapid rotation inside the cage. The ¹H NMR chemical shifts displayed a large temperature dependence owing to the paramagnetic effects.     

Two Histidines in an α‐Helix: A Rigid Co2+‐Binding Motif for PCS Measurements by NMR Spectroscopy

Pseudocontact shifts (PCS) generated by paramagnetic metal ions present valuable long‐range information in the study of protein structural biology by nuclear magnetic resonance (NMR) spectroscopy. Faithful interpretation of PCSs, however, requires complete immobilization of the metal ion relative to the protein, which is difficult to achieve with synthetic metal tags. We show that two histidine residues in sequential turns of an α‐helix provide a binding site for a Co²⁺ ion, which positions the metal ion in a uniquely well‐defined and predictable location. Exchange between the bound and free cobalt is slow on the timescale defined by chemical shifts, but the NMR resonance assignments are nonetheless readily transferred from the diamagnetic to the paramagnetic NMR spectrum by an I_zS_z‐exchange experiment. The double‐histidine‐Co²⁺ motif offers a straightforward, inexpensive, and convenient way of generating precision PCSs in proteins.     

Paramagnetic DOSY: An Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins

Diffusion ordered NMR is implemented to determine accurately the mobility of paramagnetic tris‐dipicolinate lanthanide complexes that are versatile probes of protein structure. It is shown that diffusion coefficient ratios can be measured with an accuracy of 1 % using a standard BPPLED pulse sequence, which allows for observing significant, though weak, variations when different species are interacting with the paramagnetic compound. We demonstrate that this approach is complementary to classical chemical shift titration experiments, and that it can be applied successfully to probe the supramolecular dynamic interactions between lanthanide complexes and small molecules on the one hand, or to determine rapidly their affinity for a targeted protein.     

Site‐Specific Labeling of Proteins with a Chemically Stable,High‐Affinity Tag for Protein Study

Site‐specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site‐specific manner with a high‐affinity and chemically stable tag, 4‐vinyl(pyridine‐2,6‐diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins.     

Oxygen as a paramagnetic probe of clustering and solvent exposure in folded and unfolded states of an SH3 domain

The N-terminal SH3 domain of the Drosophila modular protein Drk undergoes slow exchange between a folded (Fexch) and highly populated unfolded (Uexch) state under nondenaturing buffer conditions, enabling both Fexch and Uexch states to be simultaneously monitored. The addition of dissolved oxygen, equilibrated to a partial pressure of either 30 atm or 60 atm, provides the means to study solvent exposure with atomic resolution via 13C NMR paramagnetic shifts in 1H,13C HSQC (heteronuclear single quantum coherence) spectra. Absolute differences in these paramagnetic shifts between the Fexch and Uexch states allow the discrimination of regions of the protein which undergo change in solvent exposure upon unfolding. Contact with dissolved oxygen for both the Fexch and Uexch states could also be assessed through 13C paramagnetic shifts which were normalized based on the corresponding paramagnetic shifts seen in the free amino acids. In the Fexch state, the 13C nuclei belonging to the hydrophobic core of the protein exhibited very weak normalized paramagnetic shifts while those with greater solvent accessible surface area exhibited significantly larger normalized shifts. The Uexch state displayed less varied 13C paramagnetic shifts although distinct regions of protection from solvent exposure could be identified by a lack of such shifts. These regions, which included Phe9, Thr12, Ala13, Lys21, Thr22, Ile24, Ile27, and Arg38, overlapped with those found to have residual nativelike and non-native structures in previous studies and in some cases provided novel information. Thus, the paramagnetic shifts from dissolved oxygen are highly useful in the study of a transient structure or clustering in disordered systems, where conventional NMR measurements (couplings, chemical shift deviations from random coil values, and NOEs) may give little information.     

A Bioresistant Nitroxide Spin Label for In‐Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies

Approaching protein structural dynamics and protein–protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site‐directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide‐based spin labels for in‐cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido‐proxyl‐based spin label (M‐TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M‐TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.     

A versatile method for systematic conformational searches: application to CheY

A novel molecular structure prediction method, the Z Method, is described. It provides a versatile platform for the development and use of systematic, grid‐based conformational search protocols, in which statistical information (i.e., rotamers) can also be included. The Z Method generates trial structures by applying many changes of the same type to a single starting structure, thereby sampling the conformation space in an unbiased way. The method, implemented in the CHARMM program as the Z Module, is applied here to an illustrative model problem in which rigid, systematic searches are performed in a 36‐dimensional conformational space that describes the relative positions of the 10 secondary structural elements of the protein CheY. A polar hydrogen representation with an implicit solvation term (EEF1) is used to evaluate successively larger fragments of the protein generated in a hierarchical build‐up procedure. After a final refinement stage, and a total computational time of about two‐and‐a‐half CPU days on AMD Opteron processors, the prediction is within 1.56 Å of the native structure. The errors in the predicted backbone dihedral angles are found to approximately cancel. Monte Carlo and simulated annealing trials on the same or smaller versions of the problem, using the same atomic model and energy terms, are shown to result in less accurate predictions. Although the problem solved here is a limited one, the findings illustrate the utility of systematic searches with atom‐based models for macromolecular structure prediction and the importance of unbiased sampling in structure prediction methods. © 2011 Wiley Periodicals, Inc. J Comput Chem, 2011     

High Relaxivity Supramolecular Adducts Between Human‐Liver Fatty‐Acid‐Binding Protein and Amphiphilic GdIII Complexes: Structural Basis for the Design of Intracellular Targeting MRI Probes

Gadolinium complexes linked to an apolar fragment are known to be efficiently internalized into various cell types, including hepatocytes. Two lipid‐functionalized gadolinium chelates have been investigated for the targeting of the human liver fatty acid binding protein (hL‐FABP) as a means of increasing the sensitivity and specificity of intracellular‐directed MRI probes. hL‐FABP, the most abundant cytosolic lipid binding protein in hepatocytes, displays the ability to interact with multiple ligands involved in lipid signaling and is believed to be an obligate carrier to escort lipidic drugs across the cell. The interaction modes of a fatty acid and a bile acid based gadolinium complex with hL‐FABP have been characterized by relaxometric and NMR experiments in solution with close‐to‐physiological protein concentrations. We have introduced the analysis of paramagnetic‐induced protein NMR signal intensity changes as a quantitative tool for the determination of binding stoichiometry and of precise metal‐ion‐center positioning in protein–ligand supramolecular adducts. A few additional NMR‐derived restraints were then sufficient to locate the ligand molecules in the protein binding sites by using a rapid data‐driven docking method. Relaxometric and ¹³C NMR competition experiments with oleate and the gadolinium complexes revealed the formation of heterotypic adducts, which indicates that the amphiphilic compounds may co‐exist in the protein cavity with physiological ligands. The differences in adduct formation between fatty acid and bile acid based complexes provide the basis for an improved molecular design of intracellular targeted probes.     

In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

Self‐Association of an Enantiopure β‐Pentapeptide in Nematic Liquid Crystals

Herein, we report for the first time that nematic liquid‐crystalline environments drive the reversible self‐aggregation of an enantiopure β‐pentapeptide into oligomers with a well‐defined structure. The peptide contains four (1S,2S)‐2‐aminocyclopentane carboxylic acid (ACPC) residues and the paramagnetic β‐amino acid (3R,4R)‐4‐amino‐1‐oxyl‐2,2,5,5‐tetramethylpyrrolidine‐3‐carboxylic acid (POAC). The structure of the oligomers was investigated by electron paramagnetic resonance (EPR) spectroscopy, which allowed us to obtain the intermonomer distance distribution in the aggregates as a function of peptide concentration in two nematic liquid crystals, E7 and ZLI‐4792. The aggregates were modeled on the basis of the EPR data, and their orientation and order in the nematic phase were studied by the surface tensor method.     

Multiscale geometric modeling of macromolecules II: Lagrangian representation

Geometric modeling of biomolecules plays an essential role in the conceptualization of biolmolecular structure, function, dynamics, and transport. Qualitatively, geometric modeling offers a basis for molecular visualization, which is crucial for the understanding of molecular structure and interactions. Quantitatively, geometric modeling bridges the gap between molecular information, such as that from X‐ray, NMR, and cryo‐electron microscopy, and theoretical/mathematical models, such as molecular dynamics, the Poisson–Boltzmann equation, and the Nernst–Planck equation. In this work, we present a family of variational multiscale geometric models for macromolecular systems. Our models are able to combine multiresolution geometric modeling with multiscale electrostatic modeling in a unified variational framework. We discuss a suite of techniques for molecular surface generation, molecular surface meshing, molecular volumetric meshing, and the estimation of Hadwiger's functionals. Emphasis is given to the multiresolution representations of biomolecules and the associated multiscale electrostatic analyses as well as multiresolution curvature characterizations. The resulting fine resolution representations of a biomolecular system enable the detailed analysis of solvent–solute interaction, and ion channel dynamics, whereas our coarse resolution representations highlight the compatibility of protein‐ligand bindings and possibility of protein–protein interactions. © 2013 Wiley Periodicals, Inc.     

Reversible Photoswitching of a Spin‐Crossover Molecular Complex in the Solid State at Room Temperature

Spin‐crossover metal complexes are highly promising magnetic molecular switches for prospective molecule‐based devices. The spin‐crossover molecular photoswitches developed so far operate either at very low temperatures or in the liquid phase, which hinders practical applications. Herein, we present a molecular spin‐crossover iron(II) complex that can be switched between paramagnetic high‐spin and diamagnetic low‐spin states with light at room temperature in the solid state. The reversible photoswitching is induced by alternating irradiation with ultraviolet and visible light and proceeds at the molecular level.     

Keeping PASE with WEFT: SHWEFT–PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from ¹H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T₁ and T₂. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High‐spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one‐dimensional (1D) pulse sequences combine shaped pulses with super‐water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super‐water elimination Fourier transform–paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple ¹H 1D, 1D NOE, and two‐dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co²⁺‐amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high‐spin metBjFixLH_151–256, the heme sensing domain of Bradyrhizobium japonicum FixL. Copyright © 2013 John Wiley & Sons, Ltd.