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Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

During evolution of an RNA world, the development of enzymatic function was essential. Such enzymatic function was linked to RNA sequences capable of adopting specific RNA folds that possess catalytic pockets to promote catalysis. Within this primordial RNA world, initially evolved self-replicating ribozymes presumably mutated to ribozymes with new functions. Schultes and Bartel (Science 2000, 289, 448-452) investigated such conversion from one ribozyme to a new ribozyme with distinctly different catalytic functions. Within a neutral network that linked these two prototype ribozymes, a single RNA chain could be identified that exhibited both enzymatic functions. As commented by Schultes and Bartel, this system possessing one sequence with two enzymatic functions serves as a paradigm for an evolutionary system that allows neutral drifts by stepwise mutation from one ribozyme into a different ribozyme without loss of intermittent function. Here, we investigated this complex functional diversification of ancestral ribozymes by analyzing several RNA sequences within this neutral network between two ribozymes with class III ligase activity and with self-cleavage reactivity. We utilized rapid RNA sample preparation for NMR spectroscopic studies together with SHAPE analysis and in-line probing to characterize secondary structure changes within the neutral network. Our investigations allowed delineation of the secondary structure space and by comparison with the previously determined catalytic function allowed correlation of the structure-function relation of ribozyme function in this neutral network., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg 2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg 2+ and ligand binding., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

FK506-binding proteins (FKBPs) are promising targets for a variety of disorders and infectious diseases. High FKBP occupancy is thought to be necessary for ligands to effectively compete with the endogenous intracellular functions of FKBPs. Here, we report the development of NanoBRET assays for the most prominent cytosolic FKBPs, FKBP12, 12.6, 51 and 52. These assays allowed rapid profiling of FKBP ligands for target engagement and selectivity in living cells. These assays confirmed the selectivity of SAFit-type ligands for FKBP51 over FKBP52 but revealed a substantial offset for the intracellular activity of these ligands compared to bicyclic ligands or natural products. Our results stress the importance to control for intracellular FKBP occupancy and provide the assays to guide further FKBP ligand optimization., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The human genome contains GC-rich sequences able to form tetraplex secondary structures known as the G-quadruplex and i-motif. Such sequences are notably present in the promoter region of oncogenes and are proposed to function as regulatory elements of gene expression. The P1 promoter of BCL2 contains a 39-mer C-rich sequence (Py39wt) that can fold into a hairpin or an i-motif in a pH-dependent manner in vitro. The protein hnRNP LL was identified to recognise the i-motif over the hairpin conformation and act as an activating transcription factor. Thus, the Py39wt sequence would act as an ON/OFF switch, according to the secondary structure adopted. Herein, a structural study of the interaction between hnRNP LL and Py39wt is reported. Both N-terminal RNA recognition motifs (RRM12) cooperatively recognise one Py39wt DNA sequence and engage their β-sheet to form a large binding platform. In contrast, the C-terminal RRMs show no binding capacity. It is observed that RRM12 binds to Py39wt regardless of the DNA conformation. We propose that RRM12 recognises a single-stranded CTCCC element present in loop 1 of the i-motif and in the apical loop of the hairpin conformation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non-small-cell lung cancer). Small-molecule-based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells). 1 H, 15 N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well-folded but unstable, and it did not crystallize. However, a construct (D596-G900) produced in Sf9 cells yielded homogenous, well-folded protein that crystallized readily, thereby resulting in eleven new EPHA2-ligand crystal structures. We have also established a strategy for selective and uniform 15 N-amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2-drug interactions by NMR., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cytosine-rich single-stranded DNA oligonucleotides are able to adopt an i-motif conformation, a four-stranded structure, near a pH of 6. This unique pH-dependent conformational switch is reversible and hence can be controlled by changing the pH. Here, we show that the pH response range of the human telomeric i-motif can be shifted towards more basic pH values by introducing 5-methylcytidines (5-MeC) and towards more acidic pH values by introducing 5-bromocytidines (5-BrC). No thermal destabilisation was observed in these chemically modified i-motif sequences. The time required to attain the new conformation in response to sudden pH changes was slow for all investigated sequences but was found to be ten times faster in the 5-BrC derivative of the i-motif., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellular RNA function is closely linked to RNA structure. It is therefore imperative to develop methods that report on structural stability of RNA and how it is modulated by binding of ions, other osmolytes, and RNA-binding ligands. Here, we present a novel method to analyze the stability of virtually any structured RNA in a highly parallel fashion. This method can easily determine the influence of various additives on RNA stability, and even characterize ligand-induced stabilization of riboswitch RNA. Current approaches to assess RNA stability include thermal melting profiles (absorption or circular dichroism) and differential scanning calorimetry. These techniques, however, require a substantial amount of material and cannot be significantly parallelized. Current fluorescence spectroscopic methods rely on intercalating dyes, which alter the stability of RNA. We employ the commercial fluorescent dye RiboGreen, which discriminates between single-stranded (or unstructured regions) and double-stranded RNA. Binding leads to an increase in fluorescence quantum yield, and thus reports structural changes by a change in fluorescence intensity., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein kinases are highly dynamic and complex molecules. Here we present high-pressure and relaxation studies of the activated p38α mitogen-activated protein kinase (MAPK). p38α plays a central role in inflammatory diseases such as rheumatoid arthritis and is therefore a highly attractive pharmaceutical target. The combination of high pressure and NMR spectroscopy allowed for a detailed per-residue based assessment of the structural plasticity of p38α and the accessibility of low-lying excited-energy conformations throughout the kinase structure. Such information is uniquely accessible through the combination of liquid-state NMR and high pressure and is of considerable value for the drug discovery process. The interactions of p38α and DFG-in and DFG-out ligands were studied under the application of high pressure, and we demonstrate how we can alter kinase dynamics by pressure in a similar way to what has previously only been observed by ligand binding. Pressure is shown to be a mild and efficient tool for manipulation of intermediate-timescale dynamics., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Comparative genomics has shown that noncoding RNAs can display substantial differences between humans and chimpanzees. The human accelerated region 1 (HAR1) is a section in the human genome that exhibits the most strongly accelerated rate of nucleotide substitution in relation to the chimpanzee genome. It is associated with higher cognitive functions in human brains. The HAR1 region of the HAR1F gene is transcribed into a 118 nt noncoding RNA. We provide experimental data to validate available secondary structure models of chimpanzee and human HAR1 RNA by utilizing CD and NMR spectroscopy and applying a "divide-and-conquer" strategy. The mutations lead to more dynamic secondary and tertiary structure in the human HAR1 RNA, presumably as part of its function. We have also determined NMR solution structures of helix H1 as the most conserved part of the chimpanzee and human HAR1 RNAs. Helix H1 contains a GAA asymmetric internal loop, the structure of which had not been solved previously. 37 nt chimpanzee and human RNA fragments (c37 and h37 RNAs) differ in a single base pair. h37 RNA folds into a slightly more stable and rigid structure than c37 RNA. Both NMR structures show structural heterogeneity of the residues corresponding to the GAA loop., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Modified ubiquitin sequences, each completed with a His tag and a TEV cleavage site, were designed to enhance the expression of protein/peptide targets. With this new system we have been able to characterize several peptide-protein interactions by ITC and by NMR and CD spectroscopic methods, including the interactions of LIR domains with autophagy modifiers., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Faster than death: NMR techniques that make use of nonlinear sampling and hyperdimensional processing enable the recording of complete NMR data sets for the automated assignment of the backbone and side-chain resonances of short-lived protein samples of cell lysates., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phosphorylation of protein kinases is a central mechanism involved in numerous cellular regulatory circuits, both in prokaryotic and eukaryotic cells. An understanding of the structural and functional consequences of protein phosphorylation is of considerable importance for the design of selective, small-molecule kinase inhibitors. NMR spectroscopy is a central method to support structure-based drug design. Here, we present the NMR assignment of the activated p38α kinase and compare it to the NMR assignment of unphosphorylated p38α. Conformational changes in solution induced by activation can be located to the activation loop, an adjacent loop, and an insert part of the polypeptide chain that is specific for the family of mitogen-activated kinases. Deuterium-exchange experiments additionally revealed differences in exchange behavior for two residues in an alanine-rich helix-loop motif that becomes more flexible upon binding of an ATP analogue and a substrate peptide., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Time-resolved NMR spectroscopy was applied to study ribozyme-mediated RNA catalysis in a mutant of the hairpin ribozyme, the adenine-dependent hairpin ribozyme (ADHR; M. Meli, et al. J. Biol. Chem. 2003, 278, 9835-9842) with atomic resolution. The mutant ADHR was designed to investigate the role of cofactors in RNA catalytic mechanisms in order to understand cellular processes that could have been present in the archaic "RNA world" and of their evolution towards functional RNAs in modern cellular processes, as for example, found in the glmS ribozyme. Conformational changes due to RNA cleavage were analyzed following spectral changes of the NMR imino proton resonances that could be assigned both for the pre- and postcleaved conformation for this 80-nucleotide long RNA. (31)P NMR spectroscopic studies allowed us to confirm the formation of a cyclic phosphodiester as a result of the cleavage process. For ADHR, both metal ions and the cofactor adenine are essential for self-cleaving activity. The interaction of the ribozyme with the cofactor adenine is found to be transient and too weak to significantly change the RNA structure or to modulate the spectroscopic characteristics of the cofactor. ADHR therefore represents a ribozyme in which high activation barriers have to be overcome to populate cleavage-competent states that exhibit short life times. We show that conformational dynamics, but not the chemistry, constitute the rate-limiting step in catalysis of the adenine-dependent hairpin ribozyme.

The protein parvulustat (Z-2685) from Streptomyces parvulus comprises 78 amino acids and functions as a highly efficient alpha-amylase inhibitor. Parvulustat shares 29.6 % overall amino acid sequence identity to the well-known alpha-amylase inhibitor tendamistat. Among the conserved residues are the two disulfide bridges (C9-C25, C43-C70) and the active-site motif (W16, R17, Y18). Here, we report the high-resolution NMR structure of parvulustat based on NOEs, J couplings, chemical shifts and hydrogen-exchange data. In addition, we studied the dynamical properties of parvulustat by heteronuclear relaxation measurements. We compare the structure of parvulustat with the structure of tendamistat in terms of secondary structure elements, charges and hydrophobicity. The overall structural composition is very similar, but there are distinct differences including the active-site region. These structural and dynamical differences indicate that for parvulustat an induced-fit mechanism for binding to alpha-amylase might take place, since the structure of tendamistat does not change upon binding to alpha-amylase.

The interaction of bradykinin (BK) with the bradykinin B2 receptor (B2R) was analyzed by using molecular modeling (MM) and molecular dynamics (MD) simulations. A homology model for B2R has been generated and the recently determined receptor-bound solid-state NMR spectroscopic structure of BK (Lopez et al., Angew. Chem. 2008, 120, 1692-1695; Angew. Chem. Int. Ed. 2008, 47, 1668-1671) has been modeled into the binding pocket of the receptor to probe the putative ligand-receptor interface. The experimental hormone structure fitted well into the binding pocket of the receptor model and remained stable during the MD simulation. We propose a parallel orientation of the side chains for Arg1 and Arg9 in BK that is bound to B2R. The MD simulation study also allows the conformational changes that lead to the activated form of B2R to be analyzed. The hydrogen bond between N140 (3.35) and W283 (6.48) is the key interaction that keeps the receptor in its inactive form. This hydrogen bond is broken during the MD simulation due to rotation of transmembrane helix 3 (TM3) and is replaced by a new hydrogen bond between W283 (6.48) and N324 (7.45). We propose that this interaction is specific for the activated form of the bradykinin B2 receptor. Additionally, we compared and discussed our putative model in the context of the structural model of the partially activated rhodopsin (Rh*) and with the known biochemical and structural data.

The conformation of the cytoplasmic part of the zeta-chain of the T-cell receptor (TCR) in its free form and bound to detergent micelles has been investigated by heteronuclear NMR spectroscopy. The zeta-chain is considered to be a mediator between the extracellular antigen and the intracellular signal-transduction cascade leading to T-cell activation. Earlier studies suggested a T-cell activation mechanism that involved a TCR-state-dependent lipid incorporation propensity of the zeta-chain accompanied by a helical folding transition. In order to support this proposed mechanism, standard protein NMR assignment and secondary-structure-elucidation techniques have been applied to the free TCR zeta-chain and to the zeta-chain bound to the detergent LMPG, which forms a micelle, in order to obtain the structural characteristics of this folding transition in a residue-resolved manner. We could assign the resonances of the free zeta-chain at 278 K, and this formed the basis for chemical-shift-perturbation studies to identify lipid binding sites. Our NMR results show that the free TCR zeta-chain is indeed intrinsically unstructured. Regions around the ITAM2 and ITAM3 sequences are involved in a highly dynamic binding of the free zeta-chain to a detergent micelle formed by the acidic lipid LMPG.

Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.

Here we present an NMR-based approach to solving protein-ligand structures. The procedure is guided by biophysical, biochemical, or knowledge-based data. The structures are mainly derived from ligand-induced chemical-shift perturbations (CSP) induced in the resonances of the protein and ligand-detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS-NMR). Accuracy, as judged by comparison with X-ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical-shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein-ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein-ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.

L11, a protein of the large ribosomal subunit, binds to a highly conserved domain of 23S rRNA and mediates ribosomal GTPase activity. Its C-terminal domain is the main determinant for rRNA binding, whereas its N-terminal domain plays only a limited role in RNA binding. The N-terminal domain is thought to be involved in interactions with elongation and release factors as well as with the antibiotics thiostrepton and micrococcin. This report presents the NMR solution structure of the full-length L11 protein from the thermophilic eubacterium Thermotoga maritima in its free form. The structure is based on a large number of orientational restraints derived from residual dipolar couplings in addition to conventional NOE-based restraints. The solution structure of L11 demonstrates that, in contrast to many other multidomain RNA-binding proteins, the relative orientation of the two domains is well defined. This is shown both by heteronuclear 15N-relaxation and residual dipolar-coupling data. Comparison of this NMR structure with the X-ray structure of RNA-bound L11, reveals that binding not only induces a rigidification of a flexible loop in the C-terminal domain, but also a sizeable reorientation of the N-terminal domain. The domain orientation in free L11 shows limited similarity to that of ribosome-bound L11 in complex with elongation factor, EF-G.

This report describes NMR-spectroscopic investigations of the conformational dynamics of disulfide bonds in hen-egg-white lysozyme substitution mutants. The following four systems have been investigated: 2SS(alpha), a lysozyme variant that contains C64A, C76A, C80A and C94A substitutions, was studied in water at pH 2 and 3.8 and in urea (8 M, pH 2); 2SS(beta) lysozyme, which has C6S, C30A, C115A and C127A substitutions, was studied in water (pH 2) and urea (8 M, pH 2). The NMR analysis of heteronuclear 15N-relaxation rates shows that the barrier to disulfide-bond isomerisation can vary substantially in different lysozyme mutants and depends on the residual structure present in these states. The investigations reveal cooperativity in the modulation of micro- to millisecond dynamics that is due to the presence of multiple disulfide bridges in lysozyme. Mutation of cysteines in one of the two structural domains substantially diminishes the barrier to rotational isomerisation in the other domain. However, the interactions between hydrophobic clusters within and across the domains remains intact.

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.

NMR spectroscopy is a powerful tool for studying proteins and nucleic acids in solution. This is illustrated by the fact that nearly half of all current RNA structures were determined by using NMR techniques. Information about the structure, dynamics, and interactions with other RNA molecules, proteins, ions, and small ligands can be obtained for RNA molecules up to 100 nucleotides. This review provides insight into the resonance assignment methods that are the first and crucial step of all NMR studies, into the determination of base-pair geometry, into the examination of local and global RNA conformation, and into the detection of interaction sites of RNA. Examples of NMR investigations of RNA are given by using several different RNA molecules to illustrate the information content obtainable by NMR spectroscopy and the applicability of NMR techniques to a wide range of biologically interesting RNA molecules.