3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints

In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

CABS‐NMR—De novo tool for rapid global fold determination from chemical shifts,residual dipolar couplings and sparse methyl‐methyl noes

Recent development of nuclear magnetic resonance (NMR) techniques provided new types of structural restraints that can be successfully used in fast and low‐cost global protein fold determination. Here, we present CABS‐NMR, an efficient protein modeling tool, which takes advantage of such structural restraints. The restraints are converted from original NMR data to fit the coarse grained protein representation of the C‐Alpha‐Beta‐Side‐group (CABS) algorithm. CABS is a Monte Carlo search algorithm that uses a knowledge‐based force field. Its versatile structure enables a variety of protein‐modeling protocols, including purely de novo folding, folding guided by restraints derived from template structures or, structure assembly based on experimental data. In particular, CABS‐NMR uses the distance and angular restraints set derived from various NMR experiments. This new modeling technique was successfully tested in structure determination of 10 globular proteins of size up to 216 residues, for which sparse NMR data were available. Additional detailed analysis was performed for a S100A1 protein. Namely, we successfully predicted Nuclear Overhauser Effect signals on the basis of low‐energy structures obtained from chemical shifts by CABS‐NMR. It has been observed that utility of chemical shifts and other types of experimental data (i.e. residual dipolar couplings and methyl‐methyl Nuclear Overhauser Effect signals) in the presented modeling pipeline depends mainly on size of a protein and complexity of its topology. In this work, we have provided tools for either post‐experiment processing of various kinds of NMR data or fast and low‐cost structural analysis in the still challenging field of new fold predictions. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells

Structure determination of biomacromolecules under in‐cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site‐directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well‐suited for in‐cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in‐cell trityl–iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal‐to‐noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in‐cell measurement.     

A Reactive,Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm .     

A Reactive,Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed Gd^III spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐Gd^III is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm .     

X–H rotational-echo double-resonance NMR for torsion angle determination of peptides

C–H and N–H rotational-echo double-resonance (REDOR) NMR is developed for determining torsion angles in peptides. The distance between an X spin such as ¹³C or ¹⁵N and a proton is measured by evolving the proton magnetization under REDOR-recoupled X–H dipolar interaction. The proton of interest is selected through its directly bonded heteronuclear spin Y. The sidechain torsion angle χ₁ is extracted from a ¹³Cβ-detected Hβ–N distance, while the backbone torsion angle φ is extracted from an ¹⁵N-detected H^N–C^′ distance. The approach is demonstrated on three model peptides with known crystal structures to illustrate its utility.     

Three‐dimensional structure of cyclic antibiotic teicoplanin aglycone using NMR distance and dihedral angle restraints in a DMSO solvation model

The three‐dimensional solution conformation of teicoplanin aglycone was determined using NMR spectroscopy. A combination of NOE and dihedral angle restraints in a DMSO solvation model was used to calculate an ensemble of structures having a root mean square deviation of 0.17 Å. The structures were generated using systematic searches of conformational space for optimal satisfaction of distance and dihedral angle restraints. Comparison of the NMR‐derived structure of teicoplanin aglycone with the X‐ray structure of a teicoplanin aglycone analog revealed a common backbone conformation with deviation of two aromatic side chain substituents. Experimentally determined backbone ¹³C chemical shifts showed good agreement with those computed at the density functional level of theory, providing a cross validation of the backbone conformation. The flexible portion of the molecule was consistent with the region that changes conformation to accommodate protein binding. The results showed that a hydrogen‐bonded DMSO molecule in combination with NMR‐derived restraints together enabled calculation of structures that satisfied experimental data. Copyright © 2015 John Wiley & Sons, Ltd.     

Offset‐compensated and zero‐quantum suppressed ROESY provides accurate 1H–1H distances in small to medium‐sized molecules

A robust version of the off‐resonance ROESY pulse scheme is suggested for the measurement of proton–proton distances or slow chemical exchange in small to medium‐sized molecules. The method implements adiabatic ramps to establish a pair of opposite frequency off‐resonance spin lock fields – with optionally randomized duration – and adiabatic inversion pulses with simultaneous gradients for efficient zero‐quantum suppression. The amended pulse sequence yields pure absorption cross‐peaks and works safely for small to medium‐sized molecules. The applicability of the method has been demonstrated using small, rigid molecules (strychnine and codeine) and was also applied for a cyclic peptide and a small protein. We found that the pure phase cross‐peaks of the new ROESY version are beneficial for distance measurements. The one‐dimensional (selective) version of the new method is also powerful for measuring selected pair‐wise interactions and distance determination. Copyright © 2016 John Wiley & Sons, Ltd.     

NMR structure of a cyclic polyamide-DNA complex

The solution structure of a cyclic polyamide ligand complexed to a DNA oligomer, derived from NMR restrained molecular mechanics, is presented. The polyamide, cyclo-gamma-ImPyPy-gamma-PyPyPy-, binds to target DNA with a nanomolar dissociation constant as characterized by quantitative footprinting previously reported. 2D (1)H NMR data were used to generate distance restraints defining the structure of this cyclic polyamide with the DNA duplex d(5'-GCCTGTTAGCG-3'):d(5'-CGCTAACAGGC-3'). Data interpretation used complete relaxation matrix analysis of the NOESY cross-peak intensities with the program MARDIGRAS. The NMR-based distance restraints (276 total) were applied in restrained molecular dynamics calculations using a solvent model, yielding structures with an rmsd for the ligand and binding site of approximately 1 A. The resulting structures indicate some distortion of the DNA in the binding site. The constraints from cyclization lead to altered stacking of the rings in the halves of the cyclic ligand relative to unlinked complexes. Despite this, the interactions with DNA are very similar to what has been found in unlinked complexes. Measurements of ligand amide and DNA imino proton exchange rates indicate very slow dissociation of the ligand and show that the DNA can undergo opening fluctuations while the ligand is bound although the presence of the ligand decreases their frequency relative to the free DNA.     

Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin‐labeled in whole cells and outer membranes and interspin distances were measured to a spin‐labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein–protein/ligand interactions at surface‐exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.     

Rotamer libraries of spin labelled cysteines for protein studies

Studies of structure and dynamics of proteins using site-directed spin labelling rely on explicit modelling of spin label conformations. The large computational effort associated with such modelling with molecular dynamics (MD) simulations can be avoided by a rotamer library approach based on a coarse-grained representation of the conformational space of the spin label. We show here that libraries of about 200 rotamers, obtained by iterative projection of a long MD trajectory of the free spin label onto a set of canonical dihedral angles, provide a representation of the underlying trajectory adequate for EPR distance measurements. Rotamer analysis was performed on selected X-ray structures of spin labelled T4 lysozyme mutants to characterize the spin label rotamer ensemble on a single protein site. Furthermore, predictions based on the rotamer library approach are shown to be in nearly quantitative agreement with electron paramagnetic resonance (EPR) distance data on the Na(+)/H(+) antiporter NhaA and on the light-harvesting complex LHCII whose structures are known from independent cryo electron microscopy and X-ray studies, respectively. Suggestions for the selection of labelling sites in proteins are given, limitations of the approach discussed, and requirements for further development are outlined.     

Characterization of (1)H-(1)H distances in a uniformly (2)H,(15)N-labeled SH3 domain by MAS solid-state NMR spectroscopy (section sign)

In this communication, we demonstrate the feasibility of obtaining long-range (1)H-(1)H distance information by MAS solid-state NMR for a microcrystalline, uniformly (2)H,(15)N-labeled sample of a SH3 domain of chicken alpha-spectrin. The experiments yield NOESY-type spectra and rely on the favorable dispersion of the (15)N chemical shifts of the protein backbone. Perdeuteration of nonexchangeable sites is employed to simplify proton spin systems and to obtain multiple structural information. Two mixing schemes, (1)H-(1)H double quantum filtered Post-C7 and (1)H spin diffusion, are implemented to obtain quantitative (1)H-(1)H distance information. Post-C7 and spin diffusion cross-peak buildup rates are discussed for initial-rate fitting and in the framework of n = 0 rotational resonance (rotor driven spin diffusion), respectively. Different deuteration schemes were tested to find conditions where short-range (1)H-(1)H interactions are truncated (e.g., between H(N) and H(alpha)), but long-range interactions are retained (e.g., between H(N) and H(N)).     

Deuterated protein folds obtained directly from unassigned nuclear overhauser effect data

We demonstrate the feasibility of determining the global fold of a highly deuterated protein from unassigned experimental NMR nuclear Overhauser effect (NOE) data only. The method relies on the calculation of a spatial configuration of covalently unconnected protons-a "cloud"-directly from unassigned distance restraints derived from 13C- and 15N-edited NOESY spectra. Each proton in the cloud, labeled by its chemical shift and that of the directly bound 13C or 15N, is subsequently mapped to specific atoms in the protein. This is achieved via graph-theoretical protocols that search for connectivities in graphs that encode the structural information within the cloud. The peptidyl HN chain is traced by seeking for all possible routes and selecting the one that yields the minimal sum of sequential distances. Complete proton identification in the cloud is achieved by linking the side-chain protons to proximal main-chain HNs via bipartite graph matching. The identified protons automatically yield the NOE assignments, which in turn are used for structure calculation with RosettaNMR, a protocol that incorporates structural bias derived from protein databases. The method, named Sparse-Constraint CLOUDS, was applied to experimental NOESY data on the 58-residue Z domain of staphylococcal protein A. The generated structures are of similar accuracy to those previously reported, which were derived via a conventional approach involving a larger NMR data set. Additional tests were performed on seven reported protein structures of various folds, using restraint lists simulated from the known atomic coordinates.     

Structure calculation from unambiguous long-range amide and methyl 1H-1H distance restraints for a microcrystalline protein with MAS solid-state NMR spectroscopy

Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 ?. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j| > 5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ～1.1 ?.     

SLIM: A Short‐Linked,Highly Redox‐Stable Trityl Label for High‐Sensitivity In‐Cell EPR Distance Measurements

The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm , and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.     

Introduction of periodic boundary conditions into UNRES force field

In this article, implementation of periodic boundary conditions (PBC) into physics‐based coarse‐grained UNited RESidue (UNRES) force field is presented, which replaces droplet‐like restraints previously used. Droplet‐like restraints are necessary to keep multichain systems together and prevent them from dissolving to infinitely low concentration. As an alternative for droplet‐like restrains cuboid PBCs with imaging of the molecules were introduced. Owing to this modification, artificial forces which arose from restraints keeping a droplet together were eliminated what leads to more realistic trajectories. Due to computational reasons cutoff and smoothing functions were introduced on the long range interactions. The UNRES force field with PBC was tested by performing microcanonical simulations. Moreover, to asses the behavior of the thermostat in PBCs Langevin and Berendsen thermostats were studied. The influence of PBCs on association pattern was compared with droplet‐like restraints on the ββα hetero tetramer 1 protein system. © 2015 Wiley Periodicals, Inc.     

NMR Spin–Spin Coupling Constants in Polymethine Dyes as Polarity Indicators

Herein, we explore the use of spin–spin coupling constants (SSCCs) in merocyanine (MCYNE) dyes as indicators of polarity. For this purpose, we use Car–Parrinello hybrid quantum mechanics/molecular mechanics (QM/MM) to determine the structures of MCYNE in solvents of different polarity, followed by computations of the SSCCs by using QM/MM linear‐response theory. The molecular geometry of MCYNE switches between neutral, cyanine‐like, and zwitterionic depending on the polarity of the solvent. This structural variation is clearly reflected in the proton SSCCs in the polymethine backbone, which are highly sensitive to the dielectric nature of the environment; this mechanism can be used as a “polarity indicator” for different microenvironments. This result is highlighted by computing the SSCCs of the MCYNE probe in the cavity of the beta‐lactoglobulin protein. The computed SSCCs clearly indicate a non‐polar hydrophobic dielectric nature of this cavity.     

Direct use of unassigned resonances in NMR structure calculations with proxy residues

We present a method that significantly enhances the robustness of (automated) NMR structure determination by allowing the NOE data corresponding to unassigned NMR resonances to be used directly in the calculations. The unassigned resonances are represented by additional atoms or groups of atoms that have no interaction with the regular protein atoms except through distance restraints. These so-called "proxy" residues can be used to generate NOE-based distance restraints in a similar fashion as for the assigned part of the protein. If sufficient NOE information is available, the restraints are expected to place the proxies at positions close to the correct atoms for the unassigned resonance, which can facilitate subsequent assignment. Convergence can be further improved by supplying additional information about the possible identities of the unassigned resonances. We have implemented this approach in the widely used automated assignment and structure calculation protocols ARIA and CANDID. We find that it significantly increases the robustness of structure calculations with regard to missing assignments and yields structures of higher quality. Our approach is still able to find correctly folded structures with up to 30% randomly missing resonance assignments, and even when only backbone and beta resonances are present! This should be of significant value to NMR-based structural proteomics initiatives.