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.     

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 .     

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.     

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.     

Utilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases

Copper is an essential nutrient for the normal development of the brain and nervous system, although the hallmark of several neurological diseases is a change in copper concentrations in the brain and central nervous system. Prion protein (PrP) is a copper‐binding, cell‐surface glycoprotein that exists in two alternatively folded conformations: a normal isoform (PrP^C) and a disease‐associated isoform (PrP^Sc). Prion diseases are a group of lethal neurodegenerative disorders that develop as a result of conformational conversion of PrP^C into PrP^Sc. The pathogenic mechanism that triggers this conformational transformation with the subsequent development of prion diseases remains unclear. It has, however, been shown repeatedly that copper plays a significant functional role in the conformational conversion of prion proteins. In this review, we focus on current research that seeks to clarify the conformational changes associated with prion diseases and the role of copper in this mechanism, with emphasis on the latest applications of NMR and EPR spectroscopy to probe the interactions of copper with prion proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Thermally Activated Delayed Fluorescence in a Y3N@C80 Endohedral Fullerene: Time‐Resolved Luminescence and EPR Studies

The endohedral fullerene Y₃N@C₈₀ exhibits luminescence with reasonable quantum yield and extraordinary long lifetime. By variable‐temperature steady‐state and time‐resolved luminescence spectroscopy, it is demonstrated that above 60 K the Y₃N@C₈₀ exhibits thermally activated delayed fluorescence with maximum emission at 120 K and a negligible prompt fluorescence. Below 60 K, a phosphorescence with a lifetime of 192±1 ms is observed. Spin distribution and dynamics in the triplet excited state is investigated with X‐ and W‐band EPR and ENDOR spectroscopies and DFT computations. Finally, electroluminescence of the Y₃N@C₈₀/PFO film is demonstrated opening the possibility for red‐emitting fullerene‐based organic light‐emitting diodes (OLEDs).     

Functionalization of heteropolyanions-osmium and rhenium nitrido derivatives of Keggin- and Dawson-type polyoxotungstates: synthesis, characterization and multinuclear ((183)W, (15)N) NMR, EPR, IR, and UV/Vis fingerprints

Reaction of K(10)[alpha(2)-P(2)W(17)O(61)] or K(10)[alpha(1)-P(2)W(17)O(61)] or [Bu(4)N][OsCl(4)N] in a water/methanol mixture, and subsequent precipitation with (Bu(4)N)Br provided [alpha(2)-P(2)W(17)O(61){Os(VI)N}](7-) and [alpha(1)-P(2)W(17)O(61){Os(VI)N}](7-) Dawson structures as tetrabutylammonium salts. Reactions of [(Bu(4)N)(4)][alpha-H(3)PW(11)O(39)] with either [ReCl(3)(N(2)Ph(2))(PPh(3))(2)] or [Bu(4)N][ReCl(4)N] are alternatives to the synthesis of [(Bu(4)N)(4)][alpha-PW(11)O(39){Re(VI)N}]. (183)W and (15)N NMR, EPR, IR, and UV-visible spectroscopies and cyclic voltammetry have been used to characterize these compounds and the corresponding [(Bu(4)N)(4)][alpha-PW(11)O(39){Os(VI)N}] Keggin derivative.     

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.     

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.     

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.     

Correlation between Luminescence and EPR Spectroscopy as Evidence of Ytterbium Pair Formation in Li6Ln(BO3)3:Yb3+ (Ln=Gd,Y) Borate Single Crystals

Synthesized powders and grown single crystals of nominal compositions Li₆Ln(BO₃)₃:Yb³⁺ (Ln=Y, Gd) were investigated by means of powder and single‐crystal X‐ray diffraction (XRD), as well as optical near‐IR spectroscopy in conjunction with electron paramagnetic resonance (EPR) spectroscopy. The appearance of two distinct zero‐phonon lines suggests the existence of two kinds of Yb³⁺ ions in the single crystals. The XRD results exclude the possibility of a phase transition occurring between room and low temperatures. EPR spectra of single crystals show the presence of both isolated ions and pairs of ytterbium ions substituted for Y³⁺. A strong temperature dependence of the intensity of Yb–Yb pairs resonance lines coincides with temperature dependence of emission peak at 978 nm, confirming a common origin of the defect giving rise to these spectra. Calculated from EPR spectra, the distance between pairs of Yb³⁺ is in good agreement with crystallographic ones: R=3.856 Å, R_cryst=3.849 Å.     

Characterization of Doubly Ionic Hydrogen Bonds in Protic Ionic Liquids by NMR Deuteron Quadrupole Coupling Constants: Differences to H‐bonds in Amides,Peptides, and Proteins

We present the first deuteron quadrupole coupling constants (DQCCs) for selected protic ionic liquids (PILs) measured by solid‐state NMR spectroscopy. The experimental data are supported by dispersion‐corrected density functional theory (DFT‐D3) calculations and molecular dynamics (MD) simulations. The DQCCs of the N−D bond in the triethylammonium cations are the lowest reported for deuterons in PILs, indicating strong hydrogen bonds between ions. The NMR coupling parameters are compared to those in amides, peptides, and proteins. The DQCCs show characteristic behavior with increasing interaction strength of the counterion and variation of the H‐bond motifs. We report the similar presence of the quadrupolar splitting pattern and the narrow liquid line in the NMR spectra over large temperature ranges, indicating the heterogeneous nature of PILs.     

NMR Spectroscopic Assignment of Backbone and Side‐Chain Protons in Fully Protonated Proteins: Microcrystals,Sedimented Assemblies,and Amyloid Fibrils

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid‐state NMR spectroscopy with 100–111 kHz magic‐angle spinning (MAS). The excellent resolution in the Cα‐Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα–Hα detection block was developed and applied for the sequence‐specific backbone and aliphatic side‐chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.     

Studies Related to Norway Spruce Galactoglucomannans: Chemical Synthesis,Conformation Analysis,NMR Spectroscopic Characterization,and Molecular Recognition of Model Compounds

Galactoglucomannan (GGM) is a polysaccharide mainly consisting of mannose, glucose, and galactose. GGM is the most abundant hemicellulose in the Norway spruce (Picea abies), but is also found in the cell wall of flax seeds, tobacco plants, and kiwifruit. Although several applications for GGM polysaccharides have been developed in pulp and paper manufacturing and the food and medical industries, attempts to synthesize and study distinct fragments of this polysaccharide have not been reported previously. Herein, the synthesis of one of the core trisaccharide units of GGM together with a less‐abundant tetrasaccharide fragment is described. In addition, detailed NMR spectroscopic characterization of the model compounds, comparison of the spectral data with natural GGM, investigation of the acetyl‐group migration phenomena that takes place in the polysaccharide by using small model compounds, and a binding study between the tetrasaccharide model fragment and a galactose‐binding protein (the toxin viscumin) are reported.     

Linkage Isomerism and Spin Frustration in Heterometallic Rings: Synthesis,Structural Characterization,and Magnetic and EPR Spectroscopic Studies of Cr7Ni,Cr6Ni2, and Cr7Ni2 Rings Templated About Imidazolium Cations

The synthesis and structural characterization of three heterometallic rings templated about imidazolium cations is reported. The compounds are [2,4‐DiMe‐ImidH][Cr₇Ni^IIF₈(O₂CtBu)₁₆] 1 (2,4‐DiMe‐ImidH=the cation of 2,4‐dimethylimidazole), [ImidH]₂[Cr₆Ni^II₂F₈(O₂CCtBu)₁₆] 2 (ImidH=the cation of imidazole), and [1‐Bz‐ImidH]₂ [Cr₇Ni^II₂F₉(O₂CtBu)₁₈] 3 (1‐Bz‐ImidH=the cation of 1‐benzylimidazole). The structures show the formation of octagonal arrays of metals for 1 and 2 and a nonagon of metal centers for 3 . In all cases the edges of the polygon are bridged by a single fluoride and two pivalate ligands, and the position of the divalent metal centers cannot be distinguished by X‐ray diffraction. Magnetic studies combined with EPR spectroscopy allow the characterization of the magnetic states of the compounds. In each case the exchange is antiferromagnetic with a magnetic exchange parameter J≈?5.8 cm^?1, and it is not possible to differentiate the exchange between two Cr^III centers (J_CrCr) from the exchange between a Cr^III and a Ni^II center (J_CrNi). For 2 there is evidence for the presence of at least two, possibly four, linkage isomers of the heterometallic ring, caused by the presence of two divalent metal centers in the ring. The EPR spectroscopy of 3 suggests an S=1/2 ground state of the ring and that it is likely that only one linkage isomer is present.     

Chiral diaminopyrrolic receptors for selective recognition of mannosides, part 2: a 3D view of the recognition modes by X-ray, NMR spectroscopy, and molecular modeling

The structural features of a representative set of five complexes of octyl α- and β-mannosides with some members of a new generation of chiral tripodal diaminopyrrolic receptors, namely, (R)-5 and (S)- and (R)-7, have been investigated in solution and in the solid state by a combined X-ray, NMR spectroscopy, and molecular modeling approach. In the solid state, the binding arms of the free receptors 7 delimit a cleft in which two solvent molecules are hydrogen bonded to the pyrrolic groups and to the benzenic scaffold. In a polar solvent (CD(3)CN), chemical shift and intermolecular NOE data, assisted by molecular modeling calculations, ascertained the binding modes of the interaction between the receptor and the glycoside for these complexes. Although a single binding mode was found to adequately describe the complex of the acyclic receptor 5 with the α-mannoside, for the complexes of the cyclic receptors 7 two different binding modes were required to simultaneously fit all the experimental data. In all cases, extensive binding through hydrogen bonding and CH-π interactions is responsible for the affinities measured in the same solvent. Furthermore, the binding modes closely account for the recognition preferences observed toward the anomeric glycosides and for the peculiar enantiodiscrimination properties exhibited by the chiral receptors.     

Photochemical Reactions and Photoinduced Electron‐Transfer Processes in Liquids,Frozen Solutions,and Proteins as Studied by Multifrequency Time‐Resolved EPR Spectroscopy

In this overview, modern multifrequency EPR spectroscopy, in particular at high magnetic fields, is shown to provide detailed information about structure, motional dynamics, and spin chemistry of transient radicals and radical pairs occurring in photochemical reactions. Examples discussed comprise photochemical reactions in liquid solution and light‐initiated electron transfer processes both in biomimetic donor–acceptor model systems in frozen solution or liquid crystals and in natural photosynthetic‐reaction‐center protein complexes. The transient paramagnetic states exhibit characteristic electron polarization (CIDEP) effects. They contain valuable information about structure and dynamics of the transient reaction intermediates. Moreover, they are exploited for signal enhancement. Continuous‐wave (cw) and pulsed versions of time‐resolved high‐field EPR spectroscopy, such as cw‐transient‐EPR (TREPR) and pulsed‐electron‐spin‐echo (ESE) experiments, are compared with respect to their advantages and limitations for the specific system under study. For example, W‐band (95‐GHz) TREPR spectroscopy in conjunction with a continuous‐flow system for light‐generated short‐lived transient spin‐polarized radicals of organic photoinitiators in solution was performed with a time resolution of 10 ns. The increased Boltzmann polarization at high fields even allows detection of transient radicals without CIDEP effects. This enables one to determine initial radical polarization contributions as well as radical‐addition reaction constants. Another example of the power of combined X‐band and W‐band TREPR spectroscopy is given for the complex electron‐transfer and spin dynamics of covalently linked porphyrin–quinone as well as Watson–Crick base‐paired porphyrin–dinitrobenzene donor–acceptor biomimetic model systems. Furthermore, W‐band ESE experiments on the spin‐correlated coupled radical pair in reaction centers of the purple photosynthetic bacterium Rb. sphaeroides reveal details of distance and orientation of the pair partners in their charge‐separated transient state. The results are compared with those of the ground‐state P₈₆₅Q_A. The high orientation selectivity of high‐field EPR provides single‐crystal‐like information even from disordered frozen‐solution samples. The examples given demonstrate that high‐field EPR adds substantially to the capability of ‘classical’ spectroscopic and diffraction techniques for determining structure–dynamics–function relations of biochemical systems, since transient intermediates can be observed in real time in their working states on biologically relevant time scales.