RESUMO
MOTIVATION: Computational methods to detect correlated amino acid positions in proteins have become a valuable tool to predict intra- and inter-residue protein contacts, protein structures, and effects of mutation on protein stability and function. While there are many tools and webservers to compute coevolution scoring matrices, there is no central repository of alignments and coevolution matrices for large-scale studies and pattern detection leveraging on biological and structural annotations already available in UniProt. RESULTS: We present a Python library, PyCoM, which enables users to query and analyze coevolution matrices and sequence alignments of 457 622 proteins, selected from UniProtKB/Swiss-Prot database (length ≤ 500 residues), from a precompiled coevolution matrix database (PyCoMdb). PyCoM facilitates the development of statistical analyses of residue coevolution patterns using filters on biological and structural annotations from UniProtKB/Swiss-Prot, with simple access to PyCoMdb for both novice and advanced users, supporting Jupyter Notebooks, Python scripts, and a web API access. The resource is open source and will help in generating data-driven computational models and methods to study and understand protein structures, stability, function, and design. AVAILABILITY AND IMPLEMENTATION: PyCoM code is freely available from https://github.com/scdantu/pycom and PyCoMdb and the Jupyter Notebook tutorials are freely available from https://pycom.brunel.ac.uk.
Assuntos
Proteínas , Software , Proteínas/química , Alinhamento de Sequência , Aminoácidos , Bases de Dados de ProteínasRESUMO
MOTIVATION: Molecular dynamics (MD) simulations have become routine tools for the study of protein dynamics and function. Thanks to faster GPU-based algorithms, atomistic and coarse-grained simulations are being used to explore biological functions over the microsecond timescale, yielding terabytes of data spanning multiple trajectories, thereby extracting relevant protein conformations without losing important information is often challenging. RESULTS: We present MDSubSampler, a Python library and toolkit for a posteriori subsampling of data from multiple trajectories. This toolkit provides access to uniform, random, stratified, weighted sampling, and bootstrapping sampling methods. Sampling can be performed under the constraint of preserving the original distribution of relevant geometrical properties. Possible applications include simulations post-processing, noise reduction, and structures selection for ensemble docking. AVAILABILITY AND IMPLEMENTATION: MDSubSampler is freely available at https://github.com/alepandini/MDSubSampler, along with guidance on installation and tutorials on how it can be used.
Assuntos
Algoritmos , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic ß-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of ß-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.
Assuntos
Flavonoides/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Ilhotas Pancreáticas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Flavonoides/química , Frutas/química , Humanos , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Simulação de Dinâmica Molecular , Substâncias Protetoras/síntese química , Substâncias Protetoras/química , Agregados Proteicos/efeitos dos fármacos , Syzygium/químicaRESUMO
ß-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across ß-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.
Assuntos
COVID-19 , Peptídeo Hidrolases , Antivirais , Sítios de Ligação , Humanos , Ligantes , Inibidores de Proteases , RNA Viral , SARS-CoV-2RESUMO
Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Arabidopsis/genética , Aprendizado Profundo , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicosiltransferases/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with ß-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic ß-cells. Furthermore, AZD supplementation in pancreatic ß-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.
Assuntos
Amiloide , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Limoninas/farmacologia , Simulação de Dinâmica Molecular , Estresse Oxidativo/efeitos dos fármacos , Amiloide/química , Amiloide/metabolismo , Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Amiloidose/patologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismoRESUMO
The histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Histonas/química , Simulação de Dinâmica Molecular , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Domínios Proteicos , Estabilidade Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SolubilidadeRESUMO
Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state.
Assuntos
DNA Cruciforme/química , DNA Cruciforme/metabolismo , Resolvases de Junção Holliday/química , Resolvases de Junção Holliday/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Domínio Catalítico , Clivagem do DNA , Reparo do DNA , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Difração de Raios XRESUMO
FtsZ monomers assemble to form a dynamic Z-ring at the midcell position in bacteria that coordinates bacterial cell division. Antibacterial agents plumbagin and SB-RA-2001 were found to bind to FtsZ and to inhibit Z-ring formation in bacteria. Docking analysis indicated similar binding regions for these two inhibitors on FtsZ, and residue R191 was involved in the binding interaction with both compounds. In this work, the importance of R191 in FtsZ assembly and in bacterial cell division was analyzed. R191A-FtsZ exhibited significantly poorer polymerization ability. Further, the mutant FtsZ could poison the assembly of wild-type FtsZ (WT-FtsZ). The expression of R191A-FtsZ in Bacillus subtilis strain PL2084 perturbed Z-ring formation and produced filamentous cells, indicating that the mutation hindered the division of these cells. The results suggested that the R191A mutation is a dominant negative mutation of FtsZ. Molecular dynamics simulations of R191A-FtsZ and WT-FtsZ revealed a kink in helices H5 and H7 in the active site of R191A-FtsZ compared to that of WT-FtsZ, which is required for FtsZ assembly. The findings suggested that R191 is an important residue for FtsZ assembly, which can be targeted for the design of FtsZ inhibitors.
Assuntos
Substituição de Aminoácidos , Arginina/química , Bacillus subtilis/citologia , Proteínas de Bactérias/química , Citocinese , Proteínas do Citoesqueleto/química , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Dicroísmo Circular , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Polimerização , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1-Cul1-F-box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S-phase kinase-associated protein 2 (Skp2), cullin, and RING-finger proteins of this complex, but the roles of the adapter protein Skp1 and F-box domain of Skp2 have not been determined. Using sub-microsecond molecular dynamics simulations of full-length Skp1, unbound Skp2, Skp2-Cks1 (Cks1: Cyclin-dependent kinases regulatory subunit 1), Skp1-Skp2, and Skp1-Skp2-Cks1 complexes, we have elucidated the function of Skp1 and the F-box domain of Skp2. We found that the L16 loop of Skp1, which was deleted in previous X-ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C-terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F-box proteins. Furthermore, we observed that the F-box domain could rotate (5°-129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1-Skp2 and Skp1-Skp2-Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F-box domain of Skp2 and Skp1-Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates.
Assuntos
Proteínas Quinases Associadas a Fase S/química , Ubiquitina-Proteína Ligases/química , Proteínas Culina/química , Proteínas Culina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.
Assuntos
Cobre , DNA , Simulação de Dinâmica Molecular , Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , DNA/química , Conformação de Ácido Nucleico , Clivagem do DNA , DNA Catalítico/química , DNA Catalítico/metabolismo , Íons/químicaRESUMO
Nitroxide labels are combined with nucleic acid structures and are studied using electron paramagnetic resonance experiments (EPR). As X-ray/NMR structures are unavailable with the nitroxide labels, detailed residue level information, down to atomic resolution, about the effect of these nitroxide labels on local RNA structures is currently lacking. This information is critical to evaluate the choice of spin label. In this study, we compare and contrast the effect of TEMPO-based (NT) and rigid spin (Ç) labels (in both 2'-O methylated and not-methylated forms) on RNA duplexes. We also investigate sequence- dependent effects of NT label on RNA duplex along with the more complex G-quadruplex RNA. Distances measured from molecular dynamics simulations between the two spin labels are in agreement with the EPR experimental data. To understand the effect of labelled oligonucleotides on the structure, we studied the local base pair geometries and global structure in comparison with the unlabelled structures. Based on the structural analysis, we can conclude that TEMPO-based and Ç labels do not significantly perturb the base pair arrangements of the native oligonucleotide. When experimental structures for the spin labelled DNA/RNA molecules are not available, general framework offered by the current study can be used to provide information critical to the choice of spin labels to facilitate future EPR studies.
Assuntos
Pareamento de Bases , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Simulação de Dinâmica Molecular , Oligonucleotídeos/química , RNA/química , Marcadores de SpinRESUMO
Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires' disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.
RESUMO
Selective binding to nucleic acids and, more generally, to biopolymers, very often requires at a minimum the presence of specific functionalities and precise spatial arrangement. DNA can fold into defined 3D structures upon binding to metal centers and/or lanthanides. Binding efficiency can be boosted by modified nucleosides incorporated into DNA sequences. In this work the high selectivity of modified nucleosides towards copper (II) ions, when used in the monomeric form, is unexpectedly and drastically reduced upon being covalently attached to the DNA sequence in single-site scenario. Surprisingly, such selectivity is partially retained upon non-covalent (i.e. intercalation) mixture formed by native DNA duplex and a nucleoside in the monomeric form. Exploiting the electron spin properties of such different and rich binding mode scenarios, 1D/2D pulsed EPR experiments have been used and tailored to differentiate among the different modes. An unusual correlation of dispersion of hyperfine couplings and strength of the binding mode(s) is described.
Assuntos
Cobre/química , DNA/química , Nucleosídeos/química , Sequência de Bases , Cobre/metabolismo , DNA/genética , DNA/metabolismo , Teoria da Densidade Funcional , Modelos Moleculares , Conformação Molecular , Nucleosídeos/metabolismoRESUMO
Peb4 from Campylobacter jejuni is an intertwined dimeric, periplasmic holdase, which also exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Peb4 gene deletion alters the outer membrane protein profile and impairs cellular adhesion and biofilm formation for C. jejuni. Earlier crystallographic study has proposed that the PPIase domains are flexible and might form a cradle for holding the substrate and these aspects of Peb4 were explored using sub-microsecond molecular dynamics simulations in solution environment. Our simulations have revealed that PPIase domains are highly flexible and undergo a large structural change where they move apart from each other by 8 nm starting at .5 nm. Further, this large conformational change renders Peb4 as a compact protein with crossed-over conformation, forms a central cavity, which can "cradle" the target substrate. As reported for other chaperone proteins, flexibility of linker region connecting the chaperone and PPIase domains is key to forming the "crossed-over" conformation. The conformational transition of the Peb4 protein from the X-ray structure to the crossed-over conformation follows the "mother's arms" chain model proposed for the FkpA chaperone protein. Our results offer insights into how Peb4 and similar chaperones can use the conformational heterogeneity at their disposal to perform its much-revered biological function.
Assuntos
Proteínas de Bactérias/química , Campylobacter jejuni/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Chaperonas Moleculares/química , Peptidilprolil Isomerase/química , Fatores de Virulência/química , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Homologia Estrutural de Proteína , Especificidade por Substrato , Termodinâmica , Fatores de Virulência/metabolismoRESUMO
Ubiquitination of proteins is required to regulate several cellular mechanisms in cells. Skp1-Cullin-1-F-box (SCF), the largest family of the RING E3 ligases, recognizes and carries out the poly-ubiquitination of many substrate proteins. SCF E3 ligase is a multi-component protein complex, and the human S-phase kinase-associated protein 1 (Skp1) is the adapter protein, which binds and presents the substrate binding protein F-box (FBP) to the rest of the E3 ligase. Several crystallographic studies have solved the partial structure of Skp1 in complex with various FBPs, but there is no structure of standalone Skp1. Understanding the conformational and structural properties of Skp1 with and without FBPs is required to understand the complete mechanism of poly-ubiquitination. Here, we report ~90 % backbone and 64 % side chain (1)H, (13)C, (15)N assignments of Skp1 protein using various double and triple resonance NMR experiments.