RESUMO
In solution NMR, chemical shift perturbation (CSP) experiments are widely employed to study intermolecular interactions. However, excluding the nonsignificant peak shift is difficult because little is known about errors in CSP. Here, to address this issue, we introduce a method for estimating errors in CSP based on the noise level. First, we developed a technique that involves line shape fitting to estimate errors in peak position via Monte Carlo simulations. Second, this technique was applied to estimate errors in CSP. In intermolecular interaction analysis of VAP-A with SNX2, error estimation of CSP enabled the evaluation of small but significant changes in peak position and yielded detailed insights that are unattainable with conventional CSP analysis. Third, this technique was successfully applied to estimate errors in residual dipolar couplings. In conclusion, our error estimation method improves CSP analysis by excluding the nonsignificant peak shift.
Assuntos
Método de Monte Carlo , Nexinas de Classificação/química , Ressonância Magnética Nuclear Biomolecular , Espectroscopia de Ressonância Magnética/métodosRESUMO
NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.
Assuntos
Duplicação Gênica , Proteínas NLR , Oryza , Proteínas de Plantas , Proteínas NLR/genética , Proteínas NLR/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Evolução Molecular , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Morte Celular , Filogenia , Regulação da Expressão Gênica de PlantasRESUMO
Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF ß-sheets during the unfolding of the TTR monomer by stabilizing the interaction between ß-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.
Assuntos
Simulação de Dinâmica Molecular , Pré-Albumina , Conformação Proteica , Ligação de Hidrogênio , Pré-Albumina/química , Pré-Albumina/genética , Pré-Albumina/metabolismo , Amiloide/química , Amiloide/metabolismoRESUMO
The Biological Magnetic Resonance Data Bank (BMRB, https://bmrb.io) is the international open data repository for biomolecular nuclear magnetic resonance (NMR) data. Comprised of both empirical and derived data, BMRB has applications in the study of biomacromolecular structure and dynamics, biomolecular interactions, drug discovery, intrinsically disordered proteins, natural products, biomarkers, and metabolomics. Advances including GHz-class NMR instruments, national and trans-national NMR cyberinfrastructure, hybrid structural biology methods and machine learning are driving increases in the amount, type, and applications of NMR data in the biosciences. BMRB is a Core Archive and member of the World-wide Protein Data Bank (wwPDB).
Assuntos
Bases de Dados de Compostos Químicos , Espectroscopia de Ressonância Magnética , Bases de Dados de Proteínas , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
Fragment-based drug discovery (FBDD), which involves small compounds <300 Da, has been recognized as one of the most powerful tools for drug discovery. In FBDD, the affinity of hit compounds tends to be low, and the analysis of protein-compound interactions becomes difficult. In an effort to overcome such difficulty, we developed a 19F-NMR screening method optimizing a 19F chemical library focusing on highly soluble monomeric molecules. Our method was successfully applied to four proteins, including protein kinases and a membrane protein. For FKBP12, hit compounds were carefully validated by protein thermal shift analysis, 1H-15N HSQC NMR spectroscopy, and isothermal titration calorimetry to determine dissociation constants and model complex structures. It should be noted that the 1H and 19F saturation transfer difference experiments were crucial to obtaining highly precise model structures. The combination of 19F-NMR analysis and the optimized 19F chemical library enables the modeling of the complex structure made up of a weak binder and its target protein.
RESUMO
Structure-based high-throughput screening of chemical compounds that target protein-protein interactions (PPIs) is a promising technology for gaining insight into how plant development is regulated, leading to many potential agricultural applications. At present, there are no examples of using high-throughput screening to identify chemicals that target plant transcriptional complexes, some of which are responsible for regulating multiple physiological functions. Florigen, a protein encoded by FLOWERING LOCUS T (FT), was initially identified as a molecule that promotes flowering and has since been shown to regulate flowering and other developmental phenomena such as tuber formation in potato (Solanum tuberosum). FT functions as a component of the florigen activation complex (FAC) with a 14-3-3 scaffold protein and FD, a bZIP transcription factor that activates downstream gene expression. Although 14-3-3 is an important component of FAC, little is known about the function of the 14-3-3 protein itself. Here, we report the results of a high-throughput in vitro fluorescence resonance energy transfer (FRET) screening of chemical libraries that enabled us to identify small molecules capable of inhibiting FAC formation. These molecules abrogate the in vitro interaction between the 14-3-3 protein and the OsFD1 peptide, a rice (Oryza sativa) FD, by directly binding to the 14-3-3 protein. Treatment with S4, a specific hit molecule, strongly inhibited FAC activity and flowering in duckweed, tuber formation in potato, and branching in rice in a dose-dependent manner. Our results demonstrate that the high-throughput screening approach based on the three-dimensional structure of PPIs is suitable in plants. In this study, we have proposed good candidate compounds for future modification to obtain inhibitors of florigen-dependent processes through inhibition of FAC formation.
Assuntos
Florígeno , Oryza , Florígeno/metabolismo , Proteínas de Plantas/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Ensaios de Triagem em Larga Escala , Oryza/metabolismo , Regulação da Expressão Gênica de Plantas , Flores/genéticaRESUMO
Trinucleotide repeat (TNR) diseases are caused by the aberrant expansion of CXG (X = C, A, G and T) sequences in genomes. We have reported two small molecules binding to TNR, NCD, and NA, which strongly bind to CGG repeat (responsible sequence of fragile X syndrome) and CAG repeat (Huntington's disease). The NMR structure of NA binding to the CAG/CAG triad has been clarified, but the structure of NCD bound to the CGG/CGG triad remained to be addressed. We here report the structural determination of the NCD-CGG/CGG complex by NMR spectroscopy and the comparison with the NA-CAG/CAG complex. While the NCD-CGG/CGG structure shares the binding characteristics with that of the NA-CAG/CAG complex, a significant difference was found in the overall structure caused by the structural fluctuation at the ligand-bound site. The NCD-CGG/CGG complex was suggested in the equilibrium between stacked and kinked structures, although NA-CAG/CAG complex has only the stacked structures. The dynamic fluctuation of the NCD-CGG/CGG structure at the NCD-binding site suggested room for optimization in the linker structure of NCD to gain improved affinity to the CGG/CGG triad.
Assuntos
Carbamatos , Naftiridinas/química , DNA/química , Ligantes , Espectroscopia de Ressonância Magnética , Repetições de TrinucleotídeosRESUMO
In potato (Solanum tuberosum L.), 14-3-3 protein forms a protein complex with the FLOWERING LOCUS T (FT)-like protein StSP6A and the FD-like protein StFDL1 to activate potato tuber formation. Eleven 14-3-3 isoforms were reported in potato, designated as St14a-k. In this study, the crystal structure of the free form of St14f was determined at 2.5 Å resolution. Three chains were included in the asymmetric unit of the St14f free form crystal, and the structural deviation among the three chain structures was found on the C-terminal helix H and I. The St14f free form structure in solution was also investigated by nuclear magnetic resonance (NMR) residual dipolar coupling analysis, and the chain B in the crystal structure was consistent with NMR data. Compared to other crystal structures, St14f helix I exhibited a different conformation with larger B-factor values. Larger B-factor values on helix I were also found in the 14-3-3 free form structure with higher solvent contents. The mutation in St14f Helix I stabilized the complex with StFDL1. These data clearly showed that the flexibility of helix I of 14-3-3 protein plays an important role in the recognition of target protein.
Assuntos
Solanum tuberosum , Proteínas 14-3-3/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos/metabolismo , Solanum tuberosum/genéticaRESUMO
Glutathione peroxidase 4 (GPx4) behaves as an antioxidant enzyme capable of directly reducing peroxidized phospholipids within cell membranes. Recently, GPx4 has attracted attention as a target molecule for cancer therapy because it induces the immortalization of cancer cells suppressing ferroptosis. In this study, to analyze the function and structure of GPx4 by solution NMR, we performed resonance assignments of GPx4 and assigned almost all backbone 1H, 13C, and 15N resonances and most of the side chain 1H and 13C resonances. Using these assignments, the secondary structure of GPx4 was analyzed by the TALOS + program. GPx4 has six helices and seven strands. Then, the backbone dynamics were examined by the {1H}-15N heteronuclear NOE experiment. GPx4 was found to be rigid except for a short loop region. These results will provide basis for functional analysis and the first solution structure determination of GPx4.
Assuntos
Antioxidantes , Fosfolipídeos , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Estrutura Secundária de ProteínaRESUMO
The U-box type ubiquitin ligase PUB44 positively regulates pattern-triggered immunity in rice. Here, we identify PBI1, a protein that interacts with PUB44. Crystal structure analysis indicates that PBI1 forms a four-helix bundle structure. PBI1 also interacts with WRKY45, a master transcriptional activator of rice immunity, and negatively regulates its activity. PBI1 is degraded upon perception of chitin, and this is suppressed by silencing of PUB44 or expression of XopP, indicating that PBI1 degradation depends on PUB44. These data suggest that PBI1 suppresses WRKY45 activity when cells are in an unelicited state, and during chitin signaling, PUB44-mediated degradation of PBI1 leads to activation of WRKY45. In addition, chitin-induced MAP kinase activation is required for WRKY45 activation and PBI1 degradation. These results demonstrate that chitin-induced activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation and PUB44-mediated PBI1 degradation.
Assuntos
Oryza , Quitina/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/metabolismo , Doenças das Plantas , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.
Assuntos
Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Calorimetria/instrumentação , Calorimetria/métodos , Humanos , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética/instrumentação , Simulação de Dinâmica Molecular , Nanoestruturas/química , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Titulometria/instrumentação , Titulometria/métodosRESUMO
Importin α has been described as a nuclear protein transport receptor that enables proteins synthesized in the cytoplasm to translocate into the nucleus. Besides its function in nuclear transport, an increasing number of studies have examined its non-nuclear transport functions. In both nuclear transport and non-nuclear transport, a functional domain called the IBB domain (importin ß binding domain) plays a key role in regulating importin α behavior, and is a common interacting domain for multiple binding partners. However, it is not yet fully understood how the IBB domain interacts with multiple binding partners, which leads to the switching of importin α function. In this study, we have distinguished the location and propensities of amino acids important for each function of the importin α IBB domain by mapping the biochemical/physicochemical propensities of evolutionarily conserved amino acids of the IBB domain onto the structure associated with each function. We found important residues that are universally conserved for IBB functions across species and family members, in addition to those previously known, as well as residues that are presumed to be responsible for the differences in complex-forming ability among family members and for functional switching.
Assuntos
alfa Carioferinas , beta Carioferinas , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismoRESUMO
The endoplasmic reticulum transmembrane protein vesicle-associated membrane protein-associated protein (VAP) plays a central role in the formation and function of membrane contact sites (MCS) through its interactions with proteins. The major sperm protein (MSP) domain of VAP binds to a variety of sequences which are referred to as FFAT-like motifs. In this study, we investigated the interactions of eight peptides containing FFAT-like motifs with the VAP-A MSP domain (VAP-AMSP ) by solution NMR. Six of eight peptides are specifically bound to VAP-A. Furthermore, we found that the RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 has an FFAT-like motif which specifically binds to VAP-AMSP as well as other FFAT-like motifs. Our results will contribute to the discovery of new VAP interactors.
Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/química , Peptídeos/química , SARS-CoV-2/enzimologia , Proteínas de Transporte Vesicular/química , Motivos de Aminoácidos , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Ligação Proteica , SARS-CoV-2/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
13C-direct detection NMR has several advantages compared to proton detection, including a tendency to relax slower and wider chemical shift range. However, the sensitivity of 13C-direct detection is much lower than that of proton detection because of its lower gyromagnetic ratio. In addition, a virtual decoupling procedure is often performed to remove peak splitting in the 13C-direct detection axis, which further reduces the sensitivity to 1/â2. In this study, to enhance the sensitivity of 13C-direct detection experiments, we developed a HCACO-type new pulse sequence in which anti-phase (AP) and in-phase (IP) signals are acquired sequentially in a single scan. The developed experiment was tested on an amino acid (valine) and two proteins (streptococcal protein G B1 domain (GB1) and α-synuclein). The AP and IP spectra were successfully obtained in all cases. Using these spectra, IPAP virtual decoupling was performed, and peak splitting was successfully removed. The sensitivity of the experiment was increased by 1.43, 1.26 and 1.26 times for valine, GB1 and α-synuclein, respectively, compared to the conventional HCACO experiment. In addition, we developed another HCACO-type pulse sequence, where AP and IP signals are simultaneously acquired in a single FID. The sensitivity of the experiment was increased by 1.40 and 1.35 times for valine and GB1, respectively. These methods are potentially applicable to other 13C-direct detection experiments that measure one-bond correlations and will further extend the utility of the 13C-direct detection method, especially for structural analyses of intrinsically disordered proteins.
RESUMO
The in-cell NMR technique offers significant insights into the structure and function of heterologous proteins in the physiological intracellular environment at an atomic resolution. Escherichia coli (E. coli) is one of the most widely used host cells for heterologous protein expression in structural biological studies as well as for in-cell NMR studies to investigate fundamental structural characteristics and the physiochemistry of certain proteins and their intermolecular interactions under physiological conditions. However, in many cases, it is not easy to obtain well-resolved in-cell NMR spectra because the detectability and resolution of these spectra are significantly influenced by intracellular factors such as nonspecific intermolecular interactions. In this study, we re-examined the experimental parameters of E. coli in-cell NMR and found that the detectability and resolution of the NMR spectra clearly depended on the growth phase of the host cells. Furthermore, the detectability and resolution of the E. coli in-cell NMR spectra correlated with the soluble fraction amounts of the expressed target protein. These results indicate that the E. coli in-cell NMR spectrum of a target protein is a useful tool for monitoring the intracellular conditions of the host cell and for establishing the appropriate cultivation conditions for protein overexpression.
Assuntos
Escherichia coli/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Meio Ambiente , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Limite de Detecção , Espectroscopia de Ressonância Magnética/normasRESUMO
BACKGROUND: In protein crystals, flexible loops are frequently deformed by crystal contacts, whereas in solution, the large motions result in the poor convergence of such flexible loops in NMR structure determinations. We need an experimental technique to characterize the structural and dynamic properties of intrinsically flexible loops of protein molecules. METHODS: We designed an intended crystal contact-free space (CCFS) in protein crystals, and arranged the flexible loop of interest in the CCFS. The yeast Tim 21 protein was chosen as the model protein, because one of the loops (loop 2) is distorted by crystal contacts in the conventional crystal. RESULTS: Yeast Tim21 was fused to the MBP protein by a rigid α-helical linker. The space created between the two proteins was used as the CCFS. The linker length provides adjustable freedom to arrange loop 2 in the CCFS. We re-determined the NMR structure of yeast Tim21, and conducted MD simulations for comparison. Multidimensional scaling was used to visualize the conformational similarity of loop 2. We found that the crystal contact-free conformation of loop 2 is located close to the center of the ensembles of the loop 2 conformations in the NMR and MD structures. CONCLUSIONS: Loop 2 of yeast Tim21 in the CCFS adopts a representative, dominant conformation in solution. GENERAL SIGNIFICANCE: No single powerful technique is available for the characterization of flexible structures in protein molecules. NMR analyses and MD simulations provide useful, but incomplete information. CCFS crystallography offers a third route to this goal.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli , Espectroscopia de Ressonância Magnética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Simulação de Dinâmica Molecular , Movimento (Física) , Estrutura Secundária de Proteína , Eletricidade EstáticaRESUMO
Cdc37 is a protein kinase-targeting molecular chaperone, which cooperates with Hsp90 to assist the folding, assembly and maturation of various signaling kinases. It consists of three distinct domains: the N-terminal, middle, and C-terminal domain. While the middle domain is an Hsp90-binding domain, the N-terminal domain is recognized as a kinase-interacting domain. The N-terminal domain contains a well-conserved Ser residue at position 13, and the phosphorylation at this site has been shown to be a prerequisite for the interaction between Cdc37 and signaling kinases. Although the phosphorylation of Ser13 might induce some conformational change in Cdc37 molecule, little is known about the structure of the N-terminal domain of Cdc37. We examined the structural and dynamic properties of several fragment proteins corresponding to the N-terminal region of Cdc37 by circular dichroism and solution NMR spectroscopy. We found that the N-terminal domain of Cdc37 exhibits highly dynamic structure, and it exists in the equilibrium between α-helical and more disordered structures. We also found that phosphorylation at Ser13 did not significantly change the overall structure of N-terminal fragment protein of Cdc37. The results suggested that more complicated mechanisms might be necessary to explain the phosphorylation-activated interaction of Cdc37 with various kinases.
Assuntos
Proteínas de Ciclo Celular/química , Chaperoninas/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Dicroísmo Circular , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
The C-terminal Ig-domain of lamin A plays critical roles in cell function via interaction with proteins, DNA, and chromatin. Mutations in this domain are known to cause various diseases including Emery-Dreifuss muscular dystrophy (EDMD) and familial partial lipodystrophy (FPLD). Here we examined the biophysical and biochemical properties of mutant Ig-domains identified in patients with EDMD and FPLD. EDMD-related mutant Ig-domain showed decreased stability to heat and denaturant. This result was also confirmed by experiments using full-length mutant lamin A, although the decrease in melting temperature was much less than that of the mutant Ig-domain alone. The unstable EDMD Ig-domain disrupted the proper assembly of lamin A, resulting in abnormal paracrystal formation and decreased viscosity. In contrast, FPLD-related mutant Ig-domains were thermally stable, although they lost DNA binding function. Alanine substitution experiments revealed a functional domain of DNA binding in the Ig-domain. Thus, the overall biophysical property of Ig-domains is closely associated with clinical phenotype.
Assuntos
Lamina Tipo A/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Substituição de Aminoácidos , Fenômenos Biofísicos , DNA/química , DNA/metabolismo , Humanos , Técnicas In Vitro , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/genética , Lipodistrofia Parcial Familiar/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90-95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html. Supplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Espectroscopia de Ressonância Magnética , Redes Neurais de Computação , Proteínas , SoftwareRESUMO
Hammerhead ribozymes are a model system for studying molecular mechanism of RNA catalysis. Physicochemical data-driven mechanistic studies are an indispensable step towards understanding the catalysis of hammerhead ribozymes. Here we characterized a model RNA duplex with catalytically important sheared-type G12-A9 base pair and A9-G10.1 metal ion-binding motif in hammerhead ribozymes. By using high magnetic field NMR, all base proton signals, including catalytic residues, were unambiguously assigned. We further characterized structural features of this RNA molecule and found that it reflects the structural features of the A9-G10.1 motif of hammerhead ribozymes. Therefore, this RNA molecule is suitable for extracting an intrinsic physicochemical properties of catalytically important residues.