Probing Biomolecular Interactions with Paramagnetic Nuclear Magnetic Resonance Spectroscopy

Paramagnetism-assisted nuclear magnetic resonance (NMR) techniques have recently been applied to a wide variety of biomolecular systems, using sophisticated immobilization methods to attach paramagnetic probes, such as spin labels and lanthanide-chelating groups, at specific sites of the target biomolecules. This is also true in the field of carbohydrate NMR spectroscopy. NMR analysis of oligosaccharides is often precluded by peak overlap resulting from the lack of variability of local chemical structures, by the insufficiency of conformational restraints from nuclear Overhauser effect (NOE) data due to low proton density, and moreover, by the inherently flexible nature of carbohydrate chains. Paramagnetic probes attached to the reducing ends of oligosaccharides cause paramagnetic relaxation enhancements (PREs) and/or pseudocontact shifts (PCSs) resolve the peak overlap problem. These spectral perturbations can be sources of long-range atomic distance information, which complements the local conformational information derived from J couplings and NOEs. Furthermore, paramagnetic NMR approaches, in conjunction with computational methods, have opened up possibilities for the description of dynamic conformational ensembles of oligosaccharides in solution. Several applications of paramagnetic NMR techniques are presented to demonstrate their utility for characterizing the conformational dynamics of oligosaccharides and for probing the carbohydrate-recognition modes of proteins. These techniques can be applied to the characterization of transient, non-stoichiometric interactions and will contribute to the visualization of dynamic biomolecular processes involving sugar chains.

NaYF4 nanoparticles (NPs) are among the most promising hosts for lanthanides in different applications as IR-to-UV upconversion and photodynamic therapy due to their crystal electric field properties and their low phonon energy. The efficiency in such applications depends on the dispersion of the ions and their local structure in the host. Paramagnetic nuclear magnetic resonance (NMR) has been one of the main tools to perform such studies, based on the comprehensiveness of the hyperfine interaction of the rare-earth (RE)’s unpaired electrons with the observed nuclei and its effects in NMR quantities, like paramagnetic shift, line broadening, and relaxation enhancement. However, it can be challenging to resolve these effects for spin systems with large dipolar or quadrupolar interactions and site heterogeneity. We hereby address these issues for RE3+-doped α-NaYF4 NPs of small sizes and low polydispersity [ϕ ≈ 7(1) nm] with 23Na and 19F NMR employing high-resolution techniques associated with relaxation measurements. In particular, remarkable fast relaxation rates are found for the Gd3+-doped NPs which are rationalized in terms of Solomon’s relaxation mechanism enhanced by the long electronic relaxation time of the ion.

A metal ion with magnetic anisotropy in a biomolecule exerts strong paramagnetic effects on the nucleus spins of biomolecules in the NMR spectrum. The paramagnetic effects including paramagnetic relaxation enhancement (PRE), pseudocontact shift (PCS) and residual dipolar coupling (RDC) are valuable structural and dynamic restraints of biomolecules, and generation of these paramagnetic effects is a prerequisite. The susceptibility of magnetic anisotropy plays a similar role in the alignment of biomolecules to the commonly used alignment media in an external magnetic field. We summarize the strategies available for generating RDCs in proteins by paramagnetic labeling, selection of paramagnetic ions, and the factors influencing the magnitude of RDCs.

Nuclear magnetic resonance (NMR) spectroscopy provides detailed information about the structure and dynamics of proteins by exploiting the conformational dependence of the magnetic properties of certain atomic nuclei. The mapping between NMR measurements and molecular structures, however, often requires approximated descriptions based on the fitting of a number of parameters, thus reducing the quality of the information available from the experiments. To improve on this limitation, we show here that it is possible to use pseudocontact shifts and residual dipolar couplings as "exact" NMR restraints. We implement this strategy by using a replica-averaging method and illustrate its application by calculating an ensemble of structures representing the dynamics of the two-domain protein calmodulin.

Fragment-based lead discovery (FBLD) has proven fruitful during the past two decades for a variety of targets, even challenging protein–protein interaction (PPI) systems. Nuclear magnetic resonance (NMR) spectroscopy plays a vital role, from initial fragment-based screening to lead generation, because of its power to probe the intrinsically weak interactions between targets and low-molecular-weight fragments. Here, we review the NMR FBLD process from initial library construction to lead generation. We describe technical aspects regarding fragment library design, ligand- and protein-observed screening, and protein–ligand structure model generation. For weak binders, the initial hit-to-lead evolution can be guided by structural information retrieved from NMR spectroscopy, including chemical shift perturbation, transferred pseudocontact shifts, and paramagnetic relaxation enhancement. This perspective examines structure-guided optimization from weak fragment screening hits to potent leads for challenging PPI targets.

Paramagnetic effects are valuable structural restraints in study of structures, dynamics and interactions of proteins by means of NMR spectroscopy. Site-specific tagging of proteins with paramagnetic ions is generally required to achieve these paramagnetic effects. In this review, we first introduce the concept of paramagnetic effects including paramagnetic relaxation enhancement (PRE), pseudocontact shift (PCS) and residual dipolar couplings (RDCs), which are generally used in biomolecular NMR. Thereafter, strategies of site-specific paramagnetic tagging of proteins are outlined, and the paramagnetic effects are evaluated. On the basis of paramagnetic tagging methods described above, the applications of paramagnetic NMR in characterization of multi-domain protein replacement, 3D structural determination of unstable and low-abundance enzyme intermediate complex in real time condition, and 3D structural determination of proteins in live cells using PCSs are introduced. NMR optimized paramagnetic tags suitable for achieving high spatial resolution of distances determined by double electron-electron resonance (DEER) are reviewed. In the end, the applications of DEER measurement in characterization of protein structures in cells are demonstrated. Paramagnetic NMR and electron paramagnetic resonance (EPR) in characterization of proteins in cells is shown as a powerful technique in structural biology and chemical biology, and the development of biological compatible methods of protein modifications suitable for NMR and EPR applications are in high demand. In summary, paramagnetic tagging proteins are a powerful tool in elucidation of protein structures, dynamics and interactions in chemical and structural biology and it is surely to find wide applications in cells.

K. Kamienska-Trela (Ed.): Nuclear Magnetic Resonance. Volume 40. Specialist Periodical Reports of the Royal Society of Chemistry

1. Introduction 3721.1 Residual dipolar couplings as a route to structure and dynamics 3721.2 A brief history of oriented phase high resolution NMR 3742. Theoretical treatment of dipolar interactions 3762.1 Anisotropic interactions as probes of macromolecular structure and dynamics 3762.1.1 The dipolar interaction 3762.1.2 Averaging in the solution state 3772.2 Ordering of a rigid body 3772.2.1 The Saupe order tensor 3782.2.2 Orientational probability distribution function 3802.2.3 The generalized degree of order 3802.3 Molecular structure and internal dynamics 3813. Inducing molecular order in high resolution NMR 3833.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 3833.2 Dilute liquid crystal media: a tunable source of order 3843.2.1 Bicelles : from membrane mimics to aligning media 3853.2.2 Filamentous phage 3873.2.3 Transfer of alignment from ordered media to macromolecules 3883.3 Magnetic field alignment 3893.3.1 Paramagnetic assisted alignment 3893.3.2 Advantages of using magnetic alignment 3894. The measurement of residual dipolar couplings 3914.1 Introduction 3914.2 Frequency based methods 3924.2.1 Coupling enhanced pulse schemes 3924.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 3934.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 3954.2.4 Extraction of splitting values from the frequency domain 3964.3 Intensity based experiments 3974.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 3974.3.2 Phase modulated methods 3994.3.3 Constant time COSY – the measurement of DHH couplings 3994.3.4 Systematic errors in intensity based experiments 4005. Interpretation of residual dipolar coupling data 4015.1 Structure determination protocols utilizing orientational constraints 4015.1.1 The simulated annealing approach 4015.1.2 Order matrix analysis of dipolar couplings 4025.1.3 A discussion of the two approaches 4025.2 Reducing orientational degeneracy 4035.2.1 Multiple alignment media in the simulated annealing approach 4045.2.2 Multiple alignment media in the order matrix approach 4055.3 Simplifying effects arising due to molecular symmetry 4065.4 Database approaches for determining protein structure 4076. Applications to the characterization of macromolecular systems 4086.1 Protein structure refinement 4086.2 Protein domain orientation 4096.3 Oligosaccharides 4136.4 Biomolecular complexes 4156.5 Exchanging systems 4167. Acknowledgements 4188. References 419Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).

The detailed, quantitative characterization of unfolded proteins is a largely unresolved task due to the enormous experimental and theoretical difficulties in describing the highly dimensional space of their conformational ensembles. Recently, residual dipolar coupling (RDC) and paramagnetic relaxation enhancement (PRE) data have provided large numbers of experimental parameters on unfolded states. To obtain a minimal model of the unfolded state according to such data we have developed new modules for the use of steric alignment RDCs and PREs as constraints in ensemble structure calculations by the program XPLOR-NIH. As an example, ensemble calculations were carried out on urea-denatured ubiquitin using a total of 419 previously obtained RDCs and 253 newly determined PREs from eight cysteine mutants coupled to MTSL. The results show that only a small number of about 10 conformers is necessary to fully reproduce the experimental RDCs, PREs and average radius of gyration. C(alpha) contacts determined on a large set (400) of 10-conformer ensembles show significant (10-20%) populations of conformations that are similar to ubiquitin's A-state, i.e. corresponding to an intact native first beta-hairpin and alpha-helix as well as non-native alpha-helical conformations in the C-terminal half. Thus, methanol/acid (A-state) and urea denaturation lead to similar low energy states of the protein ensemble, presumably due to the weakening of the hydrophobic core. Similar contacts are obtained in calculations using solely RDCs or PREs. The sampling statistics of the C(alpha) contacts in the ensembles follow a simple binomial distribution. It follows that the present RDC, PRE, and computational methods allow the statistically significant detection of subconformations in the unfolded ensemble at population levels of a few percent.

A nuclear magnetic resonance advance. Imaging fluorinated anesthetics in the brain.

The determination of structural features is crucial to understand the interplay between structure and function of biomolecules and biomolecular complexes. In this context, nuclear magnetic resonance (NMR) spectroscopy provides experimental approaches, one of which is paramagnetic relaxation enhancement (PRE). Thus, placing a paramagnetic center and fluorine at strategic sites within (bio)molecules forming a complex enables the determination of distances through a straightforward, one-dimensionally guided NMR spectroscopic setup. Moreover, the almost absence of fluorine in biomolecules found in nature allows performing experimental work using cell-like or in cell conditions. Here, we made use of a single-cysteine mutant of Bacillus subtilis cold shock protein B (BsCspB) equipped with a paramagnetic spin label in complex with a fluorine-labeled variant of singly stranded DNA ligand dT4 to acquire intermolecular, 19F-based PREs. The distance between BsCspB and fluorine in dT4 has then been probed using three different experimental settings: in vitro, molecular crowding, and cell lysate conditions. Our data suggests that the intermolecular distance between the paramagnetically spin-labeled protein and the fluorine-labeled ligand does not change significantly using the three different conditions. This matches results regarding the conservation of binding affinities determined for this biomolecular complex using the three different conditions.

Nuclear magnetic resonance (NMR) spectroscopy plays a fundamental role in the stereochemical elucidation of chiral organic molecules. Together with X-ray crystallography, they belong to the mostly used methods for establishing the relative configuration. Conventional NMR methods rely on the usage of chemical shifts, nuclear Overhauser effects (NOEs), and homonuclear and heteronuclear scalar couplings ( J -couplings). In this chapter, basic theory of each NMR parameter is discussed, while several examples are demonstrated showing the power of isotropic NMR restraints in the determination of relative configuration. The application of anisotropic NMR data, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), has emerged as a powerful technique in the past 20 years for establishing constitution, relative configuration, and conformation of flexible and complex molecules. In strong contrast to NOE and scalar coupling, RDC and RCSA are long-range structural parameters. In this chapter, a number of successful examples illustrating the application of anisotropic NMR data in the determination of relative configuration will be given, where in some cases their absolute configuration could be established by a combination of anisotropic NMR and chiroptical spectroscopy. We also review recent advances in the development of new alignment media that is essential for anisotropic NMR measurements. Novel computational approaches for analyzing the anisotropic NMR parameters are briefly discussed, as these methods significantly accelerated the procedure for establishing the complete three-dimensional structures of organic molecules. In the last part, we review recent progress and application of computational NMR methods based on density functional theory (DFT) such as DP4/DP4+ and CASE-3D, which become more and more popular in determining the relative configuration of chiral organic molecules. For establishing the absolute configuration, we discuss both methods relying on chiral derivatizing agents and chiral solvents. We put a special focus on Mosher ester method based on chiral derivatization, as this method is among the most popular one. The principal and application of Mosher ester method is illustrated with published example.

Detailed knowledge of the structure of bio molecules with atomic resolution is essential for the understanding of their function. Moreover the identification and quantification of their dynamic processes are important, as they are the origin of molecular functionalities. Different spectroscopic methods like X-ray crystallography (limited to structure elucidation, no dynamics), mass spectroscopy, electron spin resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy can deliver this detailed structural information. Especially NMR spectroscopy has found its application in the identification of dynamic processes. This thesis was focused on the characterization of dynamic processes, the structure elucidation of natural products available in nanograms and the synthesis of lanthanide chelating tags for the study of protein-ligand and protein-protein interactions in solution. The thesis is divided into three chapters addressing a specific task of the mentioned topic. A: The influences of different donors and linkers on lanthanide chelating tags were investigated. A 4S-Tetramethylcyclen was used as enantiomerically pure core molecule. The side chains were based on enantiopure lactic acid derivatives. The carboxylic acids of lactic acid were transformed into different functional groups like thiols, nitrogens, and carbonyl. These different donor groups are expected to change the magnetic susceptibility tensor of the lanthanide cation in complex with the synthesized tags. Special protection protocols for the introduction of heteroatoms are presented. On this basis also two new linkers were introduced. These linkers bind in a selective way to cysteins on a protein surface as thioethers. The alpha-bromoketones are very selective for the coupling to cysteins, nevertheless they have a tendency to hydrolyse under Lewis acidic conditions. Vinylsulfones require harsher tagging conditions but they are much more stable against hydrolysis. All new tags were attached to proteins and tested as PCS reagents in protein NMR spectroscopy. B: The effects of para-substituents on the rotation barrier of 2,2«-propyl and 2,2«-butyl-bridged biphenyls were studied by dynamic NMR spectroscopy and dynamic HPLC measurements. Gibbs free activation energies of the rotation about the central biphenyl bond were estimated by variable temperature 1H-NMR experiments for the propyl bridged biphenyl. The resulting data were correlated to Hammett-parameters ?P as a measure of electron donor and acceptor strength. It was demonstrated that the electronic effects influence the activation barrier significantly, whereas sterics had only minor influence. Rate constants were calculated from line shape analysis and analysed by Eyring plots to calculate the entropic and enthalpic contributions. Thermodynamic data for the butyl-bridged biphenyls were directly obtained from dynamic HPLC chromatograms. DFT calculations delivered different transition states for the two series of biphenyls. The calculation of the activation parameters showed a similar trend and therefore the model is validated. The differences in the enthalpic and entropic contributions between HPLC, NMR and DFT calculations are method dependent, which was proven by changes of the solvent in NMR experiments that led to alteration of these contributions. C: The identification and total synthesis of a novel methylated lipid antigen (mLPA) was performed. The presented mLPA shows potent inhibition properties against human leukaemia. A combination of extraction protocols, activation essays and HPLC-MS measurements were used to identify the different antigen molecules from cell extracts. Three different candidates were identified. MS-MS experiments delivered structural insights, which were further confirmed by NMR spectroscopy and allowed the characterization of two of these structures. Total syntheses of the identified structures were performed in 6 linear steps. The high biological activity of the synthesized structures corroborated the identity of the active molecule.

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag that reacts selectively with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between lanthanoid ion and protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP), (ii) reaction of the cyano groups with α-cysteine, penicillamine or β-cysteine to complete the lanthanide chelating moiety and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-amino thiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with Gd3+ ions.

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.