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.     

一种新的稀土配合物结构分析方法-无顺磁位移混合三稀土法

提出无顺磁位移混合三稀土配合物结构分析方法, 并以丙氨酸稀土为例, 选取Eu-Yb-Pr和Er-Yb-Pr体系从实验上予以验证。研究发现, 通过选择适当的三种顺磁性稀土离子以一定比例混合, 可以抵消顺磁诱导位移, 仅保留顺磁弛豫增强效应, 因而可直观地从谱线半高宽(W1/2)看出稀土配合位点, 从纵向弛豫时间(T1)或谱线半高宽计算稀土与配体磁核间的相对距离。     

Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment

Immobilized lanthanide ions offer the opportunity to refine structures of proteins and the complexes they form by using restraints obtained from paramagnetic NMR experiments. We report the design, synthesis, and spectroscopic evaluation of the lanthanide chelator, Caged Lanthanide NMR Probe 5 (CLaNP-5) readily attachable to a protein surface via two cysteine residues. The probe causes tunable pseudocontact shifts, alignment, paramagnetic relaxation enhancement, and luminescence, by chelating it to the appropriate lanthanide ion. The observation of single shifts and the finding that the magnetic susceptibility tensors obtained from shifts and alignment analyses are highly similar strongly indicate that the probe is rigid with respect to the protein backbone. By placing the probe at various positions on a model protein it is demonstrated that the size and orientation of the magnetic susceptibility tensor of the probe are independent of the local protein environment. Consequently, the effects of the probe are readily predictable using a protein structure only. These findings designate CLaNP-5 as a protein probe to deliver unambiguous high quality structural restraints in studies on protein-protein and protein-ligand interactions.     

Beyond Bleaney's Theory: Experimental and Theoretical Analysis of Periodic Trends in Lanthanide‐Induced Chemical Shift

A detailed analysis of paramagnetic NMR shifts in a series of isostructural lanthanide complexes relavant to PARASHIFT contrast agents reveals unexpected trends in the magnetic susceptibility anisotropy that cannot be explained by the commonly used Bleaney's theory. Ab initio calculations reveal that the primary assumption of Bleaney's theory—that thermal energy is larger than the ligand field splitting—does not hold for the lanthanide complexes in question, and likely for a large fraction of lanthanide complexes in general. This makes the orientation of the magnetic susceptibility tensor differ significantly between complexes of different lanthanides with the same ligand: one of the most popular assumptions about isostructural lanthanide series is wrong.     

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.     

A justification for using NMR model-free methods when investigating the solution structures of rhombic paramagnetic lanthanide complexes

The detailed analysis of the 1H NMR hyperfine shifts according to the model-free methods shows that the semi-rigid monometallic complexes [Ln(L)(NO3)3] (Ln = Eu-Yb) are isostructural in solution. The associated separation of contact and pseudo-contact contributions to the hyperfine NMR shifts in each rhombic lanthanide complex at room temperature provides paramagnetic susceptibility tensors whose principal magnetic axes match the expected symmetry requirements. Moreover, both axial (Delta chi(ax)) and rhombic (Delta chi(rh)) paramagnetic anisotropies display satisfactory linear dependence on Bleaney's factors, a correlation predicted by the approximate high-temperature expansion of the magnetic susceptibility limited to T(-2). Consequently, the simple, and chemically attracting NMR model-free methods are not limited to axial systems, and can be safely used for the investigation of the solution structures of any lanthanide complexes. Molecular-based structural criteria for the reliable estimation of paramagnetic susceptibility tensors by NMR are discussed, together with the assignment of the labels of the crystal-field and magnetic axes within Bleaney's approach.     

3‐Mercapto‐2,6‐Pyridinedicarboxylic Acid: A Small Lanthanide‐Binding Tag for Protein Studies by NMR Spectroscopy

Paramagnetic effects from lanthanide ions present powerful tools for protein studies by nuclear magnetic resonance (NMR) spectroscopy provided that the lanthanide can be site‐specifically and rigidly attached to the protein. A new, particularly small and rigid lanthanide‐binding tag, 3‐mercapto‐2,6‐pyridinedicarboxylic acid (3MDPA), was synthesized and attached to two different proteins via a disulfide bond. The complexes of the N‐terminal domain of the E. coli arginine repressor (ArgN) with seven different paramagnetic lanthanide ions and Co²⁺ were analyzed in detail by NMR spectroscopy. The magnetic susceptibility anisotropy (Δχ) tensors and metal position were determined from pseudocontact shifts. The 3MDPA tag generated very different Δχ tensor orientations compared to the previously studied 4‐mercaptomethyl‐DPA tag, making it a highly complementary and useful tool for protein NMR studies.     

Optical absorption and NMR spectroscopic studies on paramagnetic trivalent lanthanide complexes with 2,2'-bipyridine.; The solvent effect on 4f-4f hypersensitive transitions

The optical absorption and NMR studies of trivalent lanthanide chloride complexes with 2,2'-bipyridine (bpy) are presented and discussed. The NMR spectra of paramagnetic complexes exhibit lower as well as higher field shifts of bpy resonances that reflect change in geometry and reveals importance of the factor (3 cos2 theta - 1 ) in changing sign of the shift. The paramagnetic shifts recorded have been analyzed and the intramolecular shift ratios suggest that the paramagnetic shift is predominantly dipolar in origin. Electronic spectral studies of the Pr, Nd, Ho, Er and Dy complexes in different solvents (viz. methanol, pyridine, DMSO and DMF), which differ with respect to donor atoms, reveal that the chemical environment around the lanthanide ion has great impact on f-f transitions and any change in the environment results in modifications of the spectra. The oscillator strength for the hypersensitive and non-hypersensitive transitions have been determined and changes in the oscillator strength and band shape with respect to solvent type is rationalized in terms of ligand (solvent) structure and coordination properties.     

Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix.

The full series of lanthanide ions (except the radioactive promethium and the S-state gadolinium) has been incorporated into the C-terminal calcium binding site of the dicalcium protein calbindin D(9k). A fairly constant coordination environment is maintained throughout the series. At variance with several lanthanide complexes with small chelating ligands investigated in the past, the large protein moiety provides a large number of NMR signals whose hyperfine shifts can be exclusively ascribed to pseudocontact shifts (PCS). The chemical shifts of 1H and 15N backbone and side chain amide NH groups were accurately measured through HSQC experiments. 1097 PCS were estimated from these by subtracting the diamagnetic contributions measured on HSQC spectra of either the 4f(0) lanthanum(III) or the 4f(14) lutetium(III) derivatives and used to define a quality factor for the structure. The differences in diamagnetic chemical shifts between the two diamagnetic blanks were relatively small, although some were not negligible especially for the nuclei closest to the metal center. These differences were used as a tolerance for the PCS. The magnetic susceptibility tensor anisotropies for each paramagnetic lanthanide ion were obtained as the result of the solution structure determination performed by using the NOEs of the cerium(III) derivative and the PCS of all lanthanides simultaneously. This set of reliable magnetic data permits an experimental assessment of Bleaney's theory relative to the magnetic properties for an extended series of lanthanide complexes in solution. All of the obtained tensors show some rhombicity, as could be expected from the lack of symmetry of the protein environment. The directions of the largest magnetic susceptibility component for Ce, Pr, Nd, Sm, Tb, Dy, and Ho and of the smallest magnetic susceptibility component for Eu, Er, Tm, and Yb were found to be all within 15 degrees from their average (within 20 degrees for Sm), confirming the essential similarity of the coordination environment for all lanthanides. Bleaney's theory is in excellent qualitative agreement with the observed pattern of axial anisotropies. Its quantitative agreement is substantially better than that suggested by previous analyses performed on more limited sets of PCS data for small lanthanide complexes, the so-called crystal field parameter varying only within +/-30% from one lanthanide to another. These variations are even smaller (+/-15%) if a reasonable T(-3) correction is taken into consideration. A knowledge of magnetic susceptibility anisotropy properties of lanthanides is essential in determining the self-orienting properties of lanthanide complexes in solution when immersed in magnetic fields.     

Listening to Lanthanide Complexes: Determination of the Intrinsic Luminescence Quantum Yield by Nonradiative Relaxation

The photophysical properties of lanthanide complexes have been studied extensively; however, fundamental parameters such as the intrinsic quantum yield as well as radiative and nonradiative decay rates are difficult or even impossible to measure experimentally. Herein, a photoacoustic (PA) method is proposed to determine the intrinsic quantum yield of lanthanide complexes with lifetimes in the order of milliseconds. This method is used to determine the intrinsic quantum yields for europium(III)‐containing metallomesogens as well as terbium(III) complexes. The results show that the PA signal is sensitive to both the lifetime and the ratio of the fast‐to‐slow heat component of the samples. It is found that there is an efficient ligand sensitization and a moderate intrinsic quantum yield for the complexes. The intrinsic quantum yield of Eu³⁺ in the metallomesogens exhibits an obvious increase upon the isotropic liquid to smectic A transition. The proposed PA method is quite simple, and can contribute to a clearer understanding of the photophysical processes in luminescent lanthanide complexes.     

The solution structure of rhombic lanthanide complexes analyzed with a Model-free and crystal-field independent paramagnetic NMR method: application to nonaxial trimetallic complexes [LnxLu(3-x)(TACI-3H)2(H2O)6]3+ (x = 1-3)

The model-free approach has been extended with the derivation of a novel three-nuclei crystal-field independent method for investigating isostructurality in nonaxial (i.e., rhombic) complexes along the lanthanide series. Application of this technique to the heterotrimetallic sandwich complexes [LnLu2(TACI-3H)2(H2O)6]3+, which possess a single C2v-symmetrical paramagnetic center, unambiguously evidences isostructurality for Ln = Pr-Yb, while the variation of the second-rank crystal-field parameters and along the series prevents reliable structural analyses with the classical one-nucleus equation. Extension toward polymetallic magnetically noncoupled rhombic lanthanide complexes in [Ln2Lu(TACI-3H)2(H2O)6]3+ (two paramagnetic centers with Cs microsymmetry) and [Ln3(TACI-3H)2(H2O)6]3+ (three paramagnetic centers with C2v microsymmetry) requires only minor modifications of the original three-nuclei equation. Isostructurality characterizes [Ln2Lu(TACI-3H)2(H2O)6]3+ (Ln = Pr-Yb), while [Ln3(TACI-3H)2(H2O)6]3+ exhibit a structural change between Eu and Tb which results from the concomitant contraction of the three metallic centers. Particular attention has been focused on (i) the stepwise increase of contact (i.e., through-bond) and pseudocontact (i.e., through-space) contributions when the number of paramagnetic centers increases, (ii) the assignment of 13C resonances in the strongly paramagnetic complexes [Ln3(TACI-3H)2(H2O)6]3+ (Ln = Tb-Yb) for which reliable T1 measurements and [1H-13C] correlation spectra are not accessible, and (iii) the combination of crystal-field dependent and independent methods for analyzing the paramagnetic NMR spectra of axial and nonaxial lanthanide complexes.     

Weak alignment of paramagnetic proteins warrants correction for residual CSA effects in measurements of pseudocontact shifts

Paramagnetic metal ions can induce molecular alignment with respect to the magnetic field. This alignment generates residual anisotropic chemical shifts (RACS) due to nonisotropic averaging over the molecular orientations. Using a 30 kDa protein-protein complex, the RACS effects are shown to be significant for heteronuclear spins with large chemical shift anisotropies, lanthanide ions with large anisotropic magnetic susceptibility tensors, and measurements at high magnetic field. Therefore, RACS must be taken into account when pseudocontact shifts are measured by comparison of chemical shifts observed between complexes with paramagnetic and diamagnetic lanthanide ions. The results are of particular importance when different pseudocontact shifts measured for the 1HN, 15N, and 13C' spins of a peptide group are used to restrain its orientation with respect to the electronic magnetic susceptibility tensor in structure calculations.     

Sequence-specific and stereospecific assignment of methyl groups using paramagnetic lanthanides

Pseudocontact shifts (PCSs) induced by a site-specifically bound paramagnetic lanthanide ion are shown to provide fast access to sequence-specific resonance assignments of methyl groups in proteins of known three-dimensional structure. Stereospecific assignments of Val and Leu methyls are obtained as well as resonance assignments of all other methyls, including Met epsilonCH3 groups. No prior assignments of the diamagnetic protein are required nor are experiments that transfer magnetization between the methyl groups and the protein backbone. Methyl Cz-exchange experiments were designed to provide convenient access to PCS measurements in situations where a paramagnetic lanthanide is in exchange with a diamagnetic lanthanide. In the absence of exchange, simultaneous 13C-HSQC assignments and PCS measurements are delivered by the newly developed program Possum. The approaches are demonstrated with the complex between the N-terminal domain of the subunit epsilon and the subunit theta of the Escherichia coli DNA polymerase III.     

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.     

One-dimensional simulation of lanthanide isotachophoresis using COMSOL

Electrokinetic separations can be used to quickly separate rare earth metals to determine their forensic signature. In this work, we simulate the concentration and separation of trivalent lanthanide cations by isotachophoresis. A one-dimensional simulation is developed using COMSOL v4.0a, a commercial finite element simulator, to represent the isotachophoretic separation of three lanthanides: lanthanum, terbium, and lutetium. The binding ligand chosen for complexation with the lanthanides is α-hydroxyisobutyric acid (HIBA) and the buffer system includes acetate, which also complexes with the lanthanides. The complexes formed between the three lanthanides, HIBA, and acetate are all considered in the simulation. We observe that the presence of only lanthanide:HIBA complexes in a buffer system with 10 mM HIBA causes the slowest lanthanide peak (lutetium) to split from the other analytes. The addition of lanthanide:acetate complexes into the simulation of the same buffer system eliminates this splitting. Decreasing the concentration of HIBA in the buffer to 7 mM causes the analyte stack to migrate faster through the capillary.     

4,4'-dithiobisdipicolinic acid: a small and convenient lanthanide binding tag for protein NMR spectroscopy

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structure restraints for studies of proteins by nuclear magnetic resonance spectroscopy. To elicit PCSs, the lanthanide must be attached site-specifically to the target protein. In addition, it needs to be attached rigidly to avoid averaging of the PCSs due to mobility with respect to the protein and it must not interfere with the function of the protein. Here, we present a dipicolinic acid reagent that spontaneously forms a disulfide bond with thiol groups of accessible cysteine residues. A minimal number of rotatable bonds between the cysteine side chain and the tag helps to minimise mobility. Combined with the small size of the tag and quantitative tagging yields, these features make it a highly attractive tool for generating structure restraints by paramagnetic lanthanides.     

Diamagnetic lanthanide tris β-diketonate complexes with aryl-containing ligands as chiral NMR discriminating agents

Diamagnetic lanthanum(III) and lutetium(III) tris β-diketonate complexes with the aryl-containing ligands 3-benzoyl-(+)-camphor and 3-(2-naphthoyl)-(+)-camphor are effective organic-soluble chiral NMR discriminating agents for oxygen- and nitrogen-containing compounds. Enantiomeric discrimination of sufficient magnitude to determine the enantiomeric purity is observed in the ¹H NMR spectra of compounds with hydroxyl, carbonyl, oxazolidinone, amine, and sulfoxide groups. Diamagnetic lanthanide complexes with the aryl-containing β-diketonate ligands are almost always more effective than those with 3-trifluoroacetyl-(+)-camphor, 3-heptafluorobutyryl-(+)-camphor, and d,d-dicampholylmethane that have been previously reported. Many hydrogen atoms of the substrates are significantly shielded in the presence of the lanthanide chelates with the aryl-containing ligands, which likely enhances the extent of enantiomeric discrimination in the NMR spectra. No combination of metal and ligand is most effective for all substrates. Larger enantiomeric discrimination is usually observed in benzene-d₆ or cyclohexane-d₁₂ than in chloroform-d. Diamagnetic lanthanide tris β-diketonates with the aryl-containing ligands provide an alternative to paramagnetic chelates that often cause too much broadening in the ¹H NMR spectrum.     

Lanthanide(III) complexes with a tetrapyridine pendant-armed macrocyclic ligand: 1H NMR structural determination in solution, X-ray diffraction, and density-functional theory calculations

Complexes between the tetrapyridyl pendant-armed macrocyclic ligand (L) and the trivalent lanthanide ions have been synthesized, and structural studies have been made both in the solid state and in aqueous solution. The crystal structures of the La, Ce, Pr, Gd, Tb, Er, and Tm complexes have been determined by single-crystal X-ray crystallography. In the solid state, all the cation complexes show a 10-coordinated geometry close to a distorted bicapped antiprism, with the pyridine pendants situated alternatively above and below the main plane of the macrocycle. The conformations of the two five-membered chelate rings present in the complexes change along the lanthanide series. The La(III) and Ce(III) complexes show a lambdadelta (or deltalambda) conformation, while the complexes of the heavier lanthanide ions present lambdalambda (or deltadelta) conformation. The cationic [Ln(L)]3+ complexes (Ln = La, Pr, Eu, Tb, and Tm) were also characterized by theoretical calculations at the density-functional theory (DFT) B3LYP level. The theoretical calculations predict a stabilization of the lambdalambda (or deltadelta) conformation on decreasing the ionic radius of the Ln(III) ion, in agreement with the experimental evidence. The solution structures show a good agreement with the calculated ones, as demonstrated by paramagnetic NMR measurements (lanthanide induced shifts and relaxation rate enhancements). The 1H NMR spectra indicate an effective D2 symmetry of the complexes in D2O solution. The 1H lanthanide induced shifts (LIS) observed for the Ce(III), Tm(III), and Yb(III) complexes can be fit to a theoretical model assuming that dipolar contributions are dominant for all protons. The resulting calculated values are consistent with highly rhombic magnetic susceptibility tensors with the magnetic axes being coincident with the symmetry axes of the molecule. In contrast with the solid-state structure, the analysis of the LIS data indicates that the Ce(III) complexes present a lambdalambda (or deltadelta) conformation in solution.     

Calix[4]arenes as selective extracting agents. An NMR dynamic and conformational investigation of the lanthanide(III) and thorium(IV) complexes

The lanthanide and Th4+ complexes with calix[4]arene ligands substituted either on the narrow or at the wide rim by four coordinating groups behave totally differently as shown by an NMR investigation of the dia- and paramagnetic complexes. Solutions of complexes were prepared by reacting anhydrous metal perchlorate salts with the ligands in dry acetonitrile (CAUTION). Relaxation time T1 titrations of acetonitrile solutions of Gd3+ by calixarenes indicate that ligands subsituted on the narrow rim form stable 1:1 complexes whether they feature four amide groups (1) or four phosphine oxide functions. In contrast, a ligand substituted by four (carbamoylmethyl)-diphenylphosphine oxide moieties on the wide rim (3) and its derivatives from polymeric species even at a 1:1 ligand/metal concentration ratio. Nuclear magnetic relaxation dispersion (NMRD) curves (relaxation rates 1/T1 vs magnetic field strength) of Gd3+, Gd3+.1 and Gd3+.3 perchlorates in acetonitrile are analyzed by an extended version of the Solomon-Bloembergen-Morgan equations. A comparison of the calculated rotational correlation times tau r shows that ligand 3 forms oligomeric Gd3+ species. The chelates of ligand 1 are axially symmetric (C4 symmetry), and the paramagnetic shifts induced by the Yb3+ ion are accounted for quantitatively. The addition of water or of nitrate ions does not modify the geometry of the complex. The metal chelates of 3 and its derivatives adopt a C2 symmetry, and the paramagnetic shifts are interpreted on a semiquantitative basis only. Water and NO3- ions completely labilize the complexes of the heavy lanthanides. The very high selectivity of ligand 3 through the lanthanide series stems from a complex interplay of factors.