Protein dynamics and function from solution state NMR spectroscopy.

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

Decision letter: Dynamic Na+/H+ exchanger 1 (NHE1) – calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Nuclear Magnetic Resonance (NMR) spectroscopy has become an indispensable tool in the characterization of structure and dynamics of biological macromolecules such as proteins. In recent years, NMR spectroscopy has contributed to our understanding of protein biophysics, especially when considering time-averaged dynamical events spanning various time scales. It has also played an important role in structural genomics and protein structure initiatives. The main focus of this chapter is to introduce the NMR phenomenon by providing a broad description of NMR spectroscopy and its contribution to the characterization of structure and dynamics of proteins.

Protein-ligand interactions lie at the heart of most biological and medical processes. To lay the groundwork for a mechanistic understanding of complex biological networks, a detailed description of the associated proteins, their roles, and their interaction partners is essential. Furthermore, a detailed description of protein-ligand interactions is of pharmaceutical interest, as small molecules targeting proteins constitute the basis for the treatment of numerous medical conditions. In this thesis, we used nuclear magnetic resonance (NMR) spectroscopy to probe the conformational properties of biologically and pharmaceutically essential proteins, either alone or complexed with small molecules, to reveal the mechanistic grounds of these interactions. The first chapter entails the structural investigation of Tumor necrosis factor-alpha (TNFα) by NMR spectroscopy. TNFα is a homotrimeric protein that enjoys tremendous scientific and pharmaceutical interest due to its pivotal role in the human immune response. However, its overexpression leads to numerous chronic inflammatory diseases. Studying the interaction between TNFα and small molecular inhibitors is thus of utmost importance if better cures for these diseases are to be found. Here, we investigated active and inhibited forms of TNFα in solution by NMR spectroscopy and showed that TNFα exhibits significant conformational exchange, especially in the receptor binding site and the trimer interface, which is crucial for its biological activity. Inhibition of TNFα by perturbing the trimer interface, either by mutagenesis or by targeting it with small molecules, does not lead to disruption of the postulated trimer in solution. Instead, it induces a significant increase in the conformational dynamics of TNFα propagating from the trimer interface to the receptor-binding site. Therefore, we propose that an allosteric dynamic coupling between the trimer interface and the active site mediates inhibition. This provides novel insights into the mechanisms of TNFα inhibition, which in turn may lead to the development of better-suited drug assays against this highly important molecule. The second chapter presents the study of alpha acid glycoprotein-1 (AGP1) by NMR. The heavily glycosylated AGP1 is one of the two most abundant human serum proteins and binds various metabolites as well as, perhaps most interestingly from a pharmaceutical perspective, many administered drugs. Understanding the interaction between AGP1 and small molecules on a molecular level in addition to revealing the yet unclear impact of the appended glycans in this process is thus of great importance - as drug binding to plasma proteins modulates their pharmacokinetics and pharmacodynamics. In this thesis, efficient protocols were developed which enabled us to shed light on the influence of glycosylation on the structure of AGP1 and to characterize its ligand binding at a level of detail which had not yet been achieved. We showed that glycosylation affects the protein conformation in the vicinity of the glycosylation site, and provide evidence for a conformational rearrangement upon binding of small molecules. By giving these novel insights, our results contribute to the functional understanding of AGP1 and more broadly to the relatively unexplored class of glycoproteins. The third chapter discusses the combination of our measured NMR data (presented in chapters one and two) with DNA-encoded chemical libraries (DECL) in drug discovery. DECL allows for the efficient identification of potential small molecule binders, while our concomitant NMR data provide the framework for assisting both the validation of ‘hits’ and the optimization process. We could confirm interactions between TNFα or AGP1 and binders identified by DECL. Here, the first application of this approach for drug discovery for TNFα and AGP1 is presented. In summary, our study contributes to a better understanding of two highly relevant proteins: AGP1 and TNFα. The data presented provides original insights into the inhibition mechanisms of small molecules and revealed the importance of conformational dynamics in these processes. Finally, our results will add a new aspect to the complex process of rational drug development, which will ultimately benefit many patients affected by diverse medical conditions.

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.

Chapter 1 of this thesis provides an overview of the structure and function of proteins as well as their investigation via NMR spectroscopy. Proteins are one of the most important building blocks of life. They are part of every organism and fulfill a variety of functions including, for example, the catalysis of many chemical reactions, which is the reason various processes in an organism become possible in the first place. Proteins are made up of amino acids, which are linked together via peptide bonds during the protein biosynthesis. The 22 proteinogenic amino acids result in an almost unlimited number of sequential combinations that give proteins their versatility. The sequence of amino acids in a protein is also called the primary structure. From this, locally limited structural motifs such as α-helices and β-sheets are formed, representing the secondary structure. Further intramolecular interactions create the tertiary structure of a protein, which can be seen as the overall three-dimensional structure of the protein. Furthermore, several individual proteins can assemble into a larger complex to form a quaternary structure. There are various methods for investigating the structure of proteins, including nuclear magnetic resonance (NMR) spectroscopy. In NMR spectroscopy, atomic nuclei with a non-zero nuclear spin are polarized by a constant magnetic field and then excited using radio frequency pulses. The emitted resonance frequencies are detected in the form of a free induction decay (FID) and converted into an NMR spectrum by a Fourier transformation. Many NMR experiments are available for researching proteins. By default, a two-dimensional 1H15N-HSQC spectrum is measured, which provides signals for each N-H bond in a protein meaning that one signal is obtained for each N-H in the backbone of the amino acids. Using 3D NMR experiments, these signals can then be assigned to the respective amino acids in the protein sequence. This makes it possible to investigate the dynamics of proteins as well as the formation of complexes with a ligand or another biomacromolecule if they interact. Further 3D NMR experiments also make it possible to assign the side-chains of the protein. With the help of the determined resonance assignments and spatial information from various NOESY spectra, a protein structure can then be calculated using a suitable computer program. Recently, the prediction of protein structures using AI (artificial intelligence)-supported programs has also become more prevalent. In particular, the artificial neural network AlphaFold has been extremely successful in predicting correct structures without any experimental data provided by the user. Chapter 2 of this thesis introduces the research field of small proteins. These are proteins with a length of usually less than 100 amino acids, although definitions in literature vary and upper limits of, for example, 50 or 200 amino acids exist as well. Various other names are also used to describe small proteins, such as μ-proteins or sORF-encoded peptides. The field of small proteins is still relatively new, as they were overlooked in research for many years. One reason for this was the lower limit of 100 codons that was set for the identification of open reading frames (ORFs) in genome projects. This served to exclude numerous biologically irrelevant ORFs from genome annotation. However, this meant that functional ORFs coding for small proteins were also ignored. Another reason was that experimental methods were not designed for the analysis of small proteins, meaning they could often not be detected at all. However, new analytical methods such as ribosome profiling and a generally increased focus on the detection of small proteins have led to their detection in all domains of life. In the research group of Prof. Dr. Schwalbe, small proteins of various prokaryotes were structurally characterized as part of a special priority program by the DFG. NMR spectroscopy was primarily used for this purpose, as it is the method of choice for the investigation of small proteins. The results of this research were published in several articles, two of which are part of this dissertation.

Since its invention more than six decades ago, nuclear magnetic resonance (NMR) spectroscopy has evolved as an inevitable part of chemical as well as structural analysis of small molecules, polymers, biomaterials and hybrid materials. In the solution state, due to the increased viscosity of complex viscoelastic fluids such as gels, liquid crystals and other soft materials, the rate of molecular tumbling is reduced, which in turn affects the chemical shift anisotropy, dipolar and quadrupolar interactions. As a consequence the solution state NMR spectra show broad lines, and therefore, extracting detailed structural information is a challenging task. In this context, solid state (SS) NMR has the ability to distinguish between a minute amount of polymorphic forms, conformational changes, and the number of non-equivalent molecules in an asymmetric unit of a crystal lattice, and to provide both qualitative as well as quantitative analytical data with a short-range order. Therefore, SS NMR has continued to evolve as an indispensable tool for structural analysis and gave birth to a new field called NMR crystallography. Solid state cross polarization (CP) and high resolution (HR) magic angle spinning (MAS) NMR spectroscopy has been used to study weak interactions in polymer gels. However, the application of SS NMR spectroscopy to study gels derived from low molecular weight gelators has been limited until recently. In this review, we will focus on the importance of solid state NMR spectroscopy in understanding and elucidating the structure of supramolecular gels derived from low molecular weight gelators with selected examples.

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful analytical techniques available to biology. This review is an introduction to the potential of this method and is aimed at readers who have little or no experience in acquiring or analyzing NMR spectra. We focus on spectroscopic applications of the magnetic resonance effect, rather than imaging ones, and explain how various aspects of the NMR phenomenon make it a versatile tool with which to address a number of biological problems. Using detailed examples, we discuss the use of (1) H NMR spectroscopy in mixture analysis and metabolomics, the use of (13) C NMR spectroscopy in tracking isotopomers and determining the flux through metabolic pathways ('fluxomics') and the use of (31) P NMR spectroscopy in monitoring ATP generation and intracellular pH homeotasis in vivo. Further examples demonstrate how NMR spectroscopy can be used to probe the physical environment of a cell by measuring diffusion and the tumbling rates of individual metabolites and how it can determine macromolecular structures by measuring the bonds and distances which separate individual atoms. We finish by outlining some of the key challenges which remain in NMR spectroscopy and we highlight how recent advances-such as increased magnet field strengths, cryogenic cooling, microprobes and hyperpolarisation-are opening new avenues for today's biological NMR spectroscopists.

The Biological Magnetic Resonance Data Bank (BioMagResBank or BMRB), founded in 1988, serves as the archive for data generated by nuclear magnetic resonance (NMR) spectroscopy of biological systems. NMR spectroscopy is unique among biophysical approaches in its ability to provide a broad range of atomic and higher-level information relevant to the structural, dynamic, and chemical properties of biological macromolecules, as well as report on metabolite and natural product concentrations in complex mixtures and their chemical structures. BMRB became a core member of the Worldwide Protein Data Bank (wwPDB) in 2007, and the BMRB archive is now a core archive of the wwPDB. Currently, about 10% of the structures deposited into the PDB archive are based on NMR spectroscopy. BMRB stores experimental and derived data from biomolecular NMR studies. Newer BMRB biopolymer depositions are divided about evenly between those associated with structure determinations (atomic coordinates and supporting information archived in the PDB) and those reporting experimental information on molecular dynamics, conformational transitions, ligand binding, assigned chemical shifts, or other results from NMR spectroscopy. BMRB also provides resources for NMR studies of metabolites and other small molecules that are often macromolecular ligands and/or nonstandard residues. This chapter is directed to the structural biology community rather than the metabolomics and natural products community. Our goal is to describe various BMRB services offered to structural biology researchers and how they can be accessed and utilized. These services can be classified into four main groups: (1) data deposition, (2) data retrieval, (3) data analysis, and (4) services for NMR spectroscopists and software developers. The chapter also describes the NMR-STAR data format used by BMRB and the tools provided to facilitate its use. For programmers, BMRB offers an application programming interface (API) and libraries in the Python and R languages that enable users to develop their own BMRB-based tools for data analysis, visualization, and manipulation of NMR-STAR formatted files. BMRB also provides users with direct access tools through the NMRbox platform.

The analytical potential of nuclear magnetic resonance (NMR) spectroscopy has been recognized since the early days of NMR. It is well known that the NMR signal is linear (proportional to the spin concentration), specific (spin groups have characteristic chemical shifts), and nondestructive (signal recording does not affect the sample). The last aspect is particularly important for biomedical studies, which culminated in NMR imaging (MRI) becoming an indispensable diagnostic tool. The broad application of the quantitative aspect of NMR only started after the development of NMR technology (magnets, electronics, and computers), which, in addition to selectivity, enabled the reproducibility and repeatability of data collection, even in biological systems. In this presentation, quantitative NMR microscopy and NMR spectroscopy will be illustrated by examples from studies of biological systems (live mice, fixed tissue, and body fluids). For instance, it will be shown how NMR spectroscopy can be used to quantify total proteins in biological fluids, irrespective of the protein composition, without the need for method calibration.

Chapter 27. NMR Spectroscopy in Biological Systems

Nuclear magnetic resonance (NMR) spectroscopy is a powerful experimental tool for obtaining information on three-dimensional (3D) structures of proteins at atomic resolution. In inherited forms of prion diseases, misfolding of cellular prion protein, PrPC, into its pathological form, PrPSc, is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in inherited forms of prion diseases may benefit from detailed structural analysis of different human (Hu) PrP variants. Here, we describe the protocol for structure determination of HuPrP variants by NMR spectroscopy in solution that consists of preparation of NMR samples, acquisition of NMR data, NMR resonance assignments, and structure calculation.

Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using “Backrub” motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR) Residual Dipolar Couplings (RDCs). Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i) a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii) a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics.

Chapter 13 - One- and Two-Dimensional NMR Techniques for Biopharmaceuticals

<div>Nuclear magnetic resonance (NMR) spectroscopy is one of the most</div><div>important spectroscopic methods for lignin characterization, playing a key role in its</div><div>structural elucidation and the choice of its suitable application. This chapter intends to</div><div>cover a wide range of matters regarding the NMR utilization in the lignin field from the</div><div>biorefinery up to lignin valorization through different chemical modifications in order</div><div>to produce high-added-value products with potential technological applications. The</div><div>chapter is divided into four main sections, in which the first and second ones will</div><div>provide a review of the main structural properties, NMR experiments used for lignin</div><div>characterization, and the recent advances of this technique. The third section is</div><div>composed of topics about biorefinery with a focus on microwave-assisted organosolv</div><div>delignification (MWAOD) and how the main pulping parameters affect the lignin</div><div>structure, besides the use of NMR as a quality control tool for MWAOD up-scaling.</div><div>Finally, the fourth section discusses NMR as a tool for determining structure-property</div><div>relationship by performing some chemical modifications in lignin structure aiming its</div><div>valorization, such as glycidylation, microwave-assisted phosphorylation, microwaveassisted</div><div>selective acetylation, and the performance of lignins from different pulping</div><div>processes as antioxidant compounds and the influence of their structure on it.</div><div>Therefore, this chapter contributes to the development of the lignin field by reviewing</div><div>the recent advances provided by NMR spectroscopy.</div>