Protein alignment by a coexpressed lanthanide-binding tag for the measurement of residual dipolar couplings

A protein fusion construct of human ubiquitin with an N-terminal lanthanide binding tag (LBT) enables observation of long-range orientational restraints in solution NMR from residual dipolar couplings (RDCs) due to paramagnetic alignment of the protein. The paramagnetic lanthanide ions Tb3+, Dy3+, and Tm3+ are shown to bind to the LBT and induce different alignment tensors, in agreement with theory. RDCs, measured relative to the diamagnetic Lu3+, range from -7.6 to 5.5 Hz for Tb3+ and -6.6 to 6.1 Hz for Dy3+, while an opposite alignment tensor is observed for Tm3+ (4.5 to -2.9 Hz) at 800 MHz. Experimental RDCs are in excellent agreement with those predicted on the basis of the X-ray structure of the protein.     

Lanthanide-binding peptides for NMR measurements of residual dipolar couplings and paramagnetic effects from multiple angles

Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.     

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast,Selective and reversible Paramagnetic Tagging of Proteins

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.     

Double-lanthanide-binding tags: design, photophysical properties, and NMR applications

Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.     

Rigid,Highly Reactive and Stable DOTA-like Tags Containing a Thiol-Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis**

Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.     

The 3D structure of thuricin CD, a two-component bacteriocin with cysteine sulfur to α-carbon cross-links

Thuricin CD is an antimicrobial factor that consists of two peptides, Trn-α and Trn-β, that exhibit synergistic activity against drug resistant strains of Clostridium difficile. Trn-α and Trn-β each possess three sulfur to α-carbon thioether bridges for which the stereochemistry is unknown. This report presents the three-dimensional solution structures of Trn-α and Trn-β. Structure calculations were performed for the eight possible stereoisomers of each peptide based on the same NMR data. The structure of the stereoisomer that best fit the experimental data was chosen as the representative structure for each peptide. It was determined that Trn-α has L-stereochemistry at Ser21 (α-R), L-stereochemistry at Thr25 (α-R), and D-stereochemistry at Thr28 (α-S) (an LLD isomer). Trn-β was also found to be the LLD isomer, with L-stereochemistry at Thr21 (α-R), L-stereochemistry at Ala25 (α-R), and D-stereochemistry at Tyr28 (α-S).     

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.     

Paramagnetic-based NMR restraints lift residual dipolar coupling degeneracy in multidomain detergent-solubilized membrane proteins

Residual dipolar couplings (RDCs) are widely used as orientation-dependent NMR restraints to improve the resolution of the NMR conformational ensemble of biomacromolecules and define the relative orientation of multidomain proteins and protein complexes. However, the interpretation of RDCs is complicated by the intrinsic degeneracy of analytical solutions and protein dynamics that lead to ill-defined orientations of the structural domains (ghost orientations). Here, we illustrate how restraints from paramagnetic relaxation enhancement (PRE) experiments lift the orientational ambiguity of multidomain membrane proteins solubilized in detergent micelles. We tested this approach on monomeric phospholamban (PLN), a 52-residue membrane protein, which is composed of two helical domains connected by a flexible loop. We show that the combination of classical solution NMR restraints (NOEs and dihedral angles) with RDC and PRE constraints resolves topological ambiguities, improving the convergence of the PLN structural ensemble and giving the depth of insertion of the protein within the micelle. The combination of RDCs with PREs will be necessary for improving the accuracy and precision of membrane protein conformational ensembles, where three-dimensional structures are dictated by interactions with the membrane-mimicking environment rather than compact tertiary folds common in globular proteins.     

Lanthanide-binding tags as luminescent probes for studying protein interactions

Herein, we report a method for studying protein-peptide interactions which exploits the luminescence properties of Tb(III). Lanthanide-binding tags (LBTs) are short peptide sequences comprising 15-20 naturally occurring amino acids that bind Tb(III) with high affinity. These genetically encodable luminescent tags are smaller in size than the Aequorea victoria fluorescent proteins (AFPs) and benefit from the long-lived luminescence lifetime of lanthanides. In this study, luminescence resonance energy transfer (LRET) was used to monitor the interaction between SH2 domains and different phosphopeptides. For the study, the SH2 domains of Src and Crk kinase were each coexpressed with an LBT, and phosphorylated and nonphosphorylated peptides were chemically synthesized with organic fluorophores. The LRET between the protein-bound Tb(III) and the peptide-based organic fluorophore was shown to be specific for the recognition of the SH2 domain and the peptide binding partner. This method can detect differences in binding affinity and can be used to measure the dissociation constant for the protein-peptide interaction. In addition, decay experiments can be used to calculate the distance between a site in the bound peptide and the protein using F?rster theory. In all of these experiments, the millisecond luminescence lifetime of Tb(III) can be exploited using time-resolved detection to eliminate background fluorescence from organic fluorophores.     

High-accuracy residual 1HN-13C and 1HN-1HN dipolar couplings in perdeuterated proteins

Truncation by the presence of many short-range residual dipolar couplings (RDCs) hinders the observation of long-range RDCs in weakly aligned biomacromolecules. Perdeuteration of proteins followed by reprotonation of labile hydrogen positions greatly alleviates this problem. Here we show that for small perdeuterated proteins, a large number (up to 10 in protein G) of long-range RDCs to 13C and 1HN can be observed from individual amide protons. The 1HN <--> 13C RDCs comprise correlations to 13Calpha, 13Cbeta, and 13C' nuclei of the same and the preceding amino acid, as well as 13C' nuclei of hydrogen-bonded amino acids. The accuracy of the coupling constants is very high and defines individual internuclear distances to within few picometers. Deviations between measured RDC values and values predicted from the 1.1 A crystal structure of protein G are mainly found in two surface-exposed loop regions. The deviations show a strong correlation to the B-factor of the crystal structure.     

Orientation of deoxyhemoglobin at high magnetic fields: structural insights from RDCs in solution

Human normal adult hemoglobin (Hb A) is a tetrameric protein molecule of ~64 kDa consisting of two identical -chains and two identical -chains of 141 and 146 amino acid residues each and four bound heme moieties. In the oxygen-free form of Hb A, also known as deoxyhemoglobin A (deoxy-Hb A), the hemes are paramagnetic with S = 2. We have measured the one-bond spin-spin couplings (1JNH + 1DNH) on (15N,2H)-labeled deoxy-Hb A in solution as a function of magnetic field strengths from 11.7 to 21.1 T and found that these couplings are linearly proportional to the square of the magnetic field. This field dependence provides an opportunity to extract the residual dipolar couplings (RDCs, 1DNH) and, thus, to compare predictions about the solution structure of deoxy-Hb A to crystal structures for this molecule. Such comparison is essential for our understanding of the structure, dynamics, and function of this allosteric protein under conditions close to the physiological state. This report illustrates the usefulness of using the magnetic-field dependent RDCs to determine the solution structure of a large paramagnetic protein. This method is especially valuable for those proteins whose structures must be determined in an oxygen-free environment.     

Bound-state residual dipolar couplings for rapidly exchanging ligands of His-tagged proteins

The study of bound-state conformations of ligands interacting with proteins is important to the understanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclear Overhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between bound and free states, have been used in these studies, owing to the inherent heavy weighting of bound-state data in the averaged ligand signals. In principle, residual dipolar couplings (RDCs) provide a useful complement to NOE data in that they provide orientational constraints as opposed to distance constraints, but use in ligand-binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound-state data in averaged RDCs measured on ligands is presented. The approach rests on association of a His-tagged protein with a nickel-chelate-carrying lipid inserted into the lipid bilayer-like alignment media used in the acquisition of RDCs. The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose, bound to the carbohydrate recognition domain of the mammalian lectin, galectin-3.     

Highly populated turn conformations in natively unfolded tau protein identified from residual dipolar couplings and molecular simulation

Tau, a natively unstructured protein that regulates the organization of neuronal microtubules, is also found in high concentrations in neurofibrillary tangles of Alzheimer's disease and other neurodegenerative disorders. The conformational transition between these vastly different healthy and pathological forms remains poorly understood. We have measured residual dipolar couplings (RDCs), J-couplings, and nuclear Overhauser enhancement (NOE) in construct K18 of tau, containing all four repeat domains R1-R4. NHN RDCs were compared with prediction on the basis of a statistical model describing the intrinsic conformational sampling of unfolded proteins in solution. While local variation and relative amplitude of RDCs agrees with propensity-based prediction for most of the protein, homologous sequences in each repeat domain (DLKN, DLSN, DLSK, and DKFD in repeats R1-R4) show strong disagreement characterized by inversion of the sign of the central couplings. Accelerated molecular dynamic simulations (AMD) in explicit solvent revealed strong tendencies to form turns, identified as type I beta-turns for repeats R1-R3. Incorporation of the backbone dihedral sampling resulting from AMD into the statistical coil model closely reproduces experimental RDC values. These localized sequence-dependent conformational tendencies interrupt the propensity to sample more extended conformations in adjacent strands and are remarkably resistant to local environmental factors, as demonstrated by the persistence of the RDC signature even under harsh denaturing conditions (8 M urea). The role that this specific conformational behavior may play in the transition to the pathological form is discussed.     

Stabilization of a β-Hairpin Conformation in a Cyclic Peptide Using the Templating Effect of a Heterochiral Diproline Unit

A straightforward and effective method of stabilizing a β-hairpin conformation in a cyclic protein loop mimetic is described, which exploits the templating effect of a heterochiral D -Pro-L -Pro dipeptide unit. A twelve-residue β-hairpin loop was grafted from the extracellular interferon γ receptor onto the heterochiral D -Pro-L -Pro dipeptide template to afford a fourteen-residue cyclic peptide. The residues directly attached to the D -Pro-L -Pro template are shown by NMR spectroscopy to structurally mimic corresponding residues in adjacent antiparallel β-strands in the receptor. MD Simulations with and without time-averaged distance restraints support this view and indicate that the tip of the loop is more flexible, as inferred also for the receptor protein from crystallographic data. The templating effect of the heterochiral diproline unit also promotes efficient backbone cyclization of the fourteen-residue linear peptide precursor, suggesting that a wide variety of related protein loop mimetics incorporating the D -Pro-L -Pro template might be readily accessible.     

Conformational Ensemble of Disordered Proteins Probed by Solvent Paramagnetic Relaxation Enhancement (sPRE)

Characterization of the conformational ensemble of disordered proteins is highly important for understanding protein folding and aggregation mechanisms, but remains a computational and experimental challenge owing to the dynamic nature of these proteins. New observables that can provide unique insights into transient residual structures in disordered proteins are needed. Here using denatured ubiquitin as a model system, NMR solvent paramagnetic relaxation enhancement (sPRE) measurements provide an accurate and highly sensitive probe for detecting low populations of residual structure in a disordered protein. Furthermore, a new ensemble calculation approach based on sPRE restraints in conjunction with residual dipolar couplings (RDCs) and small‐angle X‐ray scattering (SAXS) is used to define the conformational ensemble of disordered proteins at atomic resolution. The approach presented should be applicable to a wide range of dynamic macromolecules.     

Probing Long‐Range Anisotropic Interactions: a General and Sign‐Sensitive Strategy to Measure 1H–1H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination

Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one‐bond ¹H–¹³C RDCs alone often fall short. Long‐range ¹H–¹H RDCs are both abundant and typically sample highly complementary orientations, but accessing them in a sign‐sensitive way has been severely obstructed due to the overflow of ¹H–¹H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of ¹H–¹H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant‐time β‐COSY experiment. The potential of ¹H–¹H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.     

Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins

High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR. Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins. (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E. coli sulfite reductase. These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame. The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample. To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model. Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.     

Development of bifunctional stilbene derivatives for targeting and modulating metal-amyloid-β species

Amyloid-β (Aβ) peptides and their metal-associated aggregated states have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the etiology of AD remains uncertain, understanding the role of metal-Aβ species could provide insights into the onset and development of the disease. To unravel this, bifunctional small molecules that can specifically target and modulate metal-Aβ species have been developed, which could serve as suitable chemical tools for investigating metal-Aβ-associated events in AD. Through a rational structure-based design principle involving the incorporation of a metal binding site into the structure of an Aβ interacting molecule, we devised stilbene derivatives (L1-a and L1-b) and demonstrated their reactivity toward metal-Aβ species. In particular, the dual functions of compounds with different structural features (e.g., with or without a dimethylamino group) were explored by UV-vis, X-ray crystallography, high-resolution 2D NMR, and docking studies. Enhanced bifunctionality of compounds provided greater effects on metal-induced Aβ aggregation and neurotoxicity in vitro and in living cells. Mechanistic investigations of the reaction of L1-a and L1-b with Zn(2+)-Aβ species by UV-vis and 2D NMR suggest that metal chelation with ligand and/or metal-ligand interaction with the Aβ peptide may be driving factors for the observed modulation of metal-Aβ aggregation pathways. Overall, the studies presented herein demonstrate the importance of a structure-interaction-reactivity relationship for designing small molecules to target metal-Aβ species allowing for the modulation of metal-induced Aβ reactivity and neurotoxicity.     

Dipolar waves as NMR maps of protein structure

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.