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1.
J Chem Phys ; 160(10)2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38465679

RESUMO

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields.


Assuntos
Simulação de Dinâmica Molecular , Ubiquitina , Ubiquitina/química , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Espectroscopia de Ressonância Magnética
2.
Methods Mol Biol ; 2754: 271-306, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38512672

RESUMO

Posttranslational modifications (PTMs) of proteins can be investigated by Nuclear Magnetic Resonance (NMR) spectroscopy as a powerful analytical tool to define modification sites, their relative stoichiometry, and crosstalk between modifications. As a Structural Biology method, NMR provides important additional information on changes in protein conformation and dynamics upon modification as well as a mapping of binding sites upon biomolecular interactions. Indeed, PTMs not only mediate functional modulation in protein-protein interactions, but can also induce diverse structural responses with different biological outcomes. Here we present protocols that have been developed for the production and phosphorylation of the neuronal tau protein. Under its aggregated form, tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases named tauopathies involving tau dysfunction and/or mutations. As a common feature shared by various tauopathies, tau aggregates are found into a form displaying an increased, abnormal phosphorylation, also referred to hyperphosphorylation. We have used NMR to investigate the phosphorylation patterns of tau induced by several kinases or cell extracts, how phosphorylation affects the local and overall conformation of tau, its interactions with partners (proteins, DNA, small-molecules, etc.) including tubulin and microtubules, and its capacity to form insoluble fibrillar aggregates. We present here detailed protocols for in vitro phosphorylation of tau by the recombinant kinases CDK2/cyclin A and GSK3ß, the production of the recombinant kinases thereof, as well as the analytical characterization of phosphorylated tau by NMR spectroscopy.


Assuntos
Doença de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Fosforilação , Glicogênio Sintase Quinase 3 beta/metabolismo , Ciclina A/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Doença de Alzheimer/metabolismo , Espectroscopia de Ressonância Magnética , Quinase 2 Dependente de Ciclina/genética
3.
Protein Sci ; 33(4): e4950, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38511503

RESUMO

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.


Assuntos
Aminoácidos , Escherichia coli , Animais , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Mamíferos
4.
Adv Exp Med Biol ; 3234: 109-123, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507203

RESUMO

Nuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in the context of their applications to structural studies in biology with specific focus on protein interactions and evaluate their limitations. We provide specific examples in which these techniques can complement each other, providing new information on the same scientific case. We discuss how the field may develop and what challenges are expected in the future. Overall, the combination of NMR and MS plays an increasingly important role in integrative structural biology, assisting scientists in deciphering the three-dimensional structure of composite macromolecular assemblies.


Assuntos
Imageamento por Ressonância Magnética , Espectrometria de Massas/métodos , Espectroscopia de Ressonância Magnética , Substâncias Macromoleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos
5.
Protein Sci ; 33(4): e4922, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38501482

RESUMO

The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor-NIH biomolecular structure determination package. In combination with an improved treatment of selected non-bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters are tested using structure calculations on 30 proteins with restraints derived from nuclear magnetic resonance data. Using modern structure validation criteria, including several formally adopted by the Protein Data Bank, and a clear measure of structural accuracy, the results show superior performance relative to previous Xplor-NIH implementations. Additionally, the Xplor-NIH structures compare favorably against originally determined NMR models.


Assuntos
Proteínas , Software , Proteínas/química , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica
6.
J Phys Chem Lett ; 15(8): 2270-2278, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38381862

RESUMO

NMR chemical shifts provide a sensitive probe of protein structure and dynamics but remain challenging to predict and interpret. We examine the effect of protein conformational distributions on 15N chemical shifts for dihydrofolate reductase (DHFR), comparing QM/MM predicted shifts with experimental shifts in solution as well as frozen distributions. Representative snapshots from MD trajectories exhibit variation in predicted 15N chemical shifts of up to 25 ppm. The average over the fluctuations is in significantly better agreement with room temperature solution experimental values than the prediction for any single optimal conformations. Meanwhile, solid-state NMR (SSNMR) measurements of frozen solutions at 105 K exhibit broad lines whose widths agree well with the widths of distributions of predicted shifts for samples from the trajectory. The backbone torsion angle ψi-1 varies over 60° on the picosecond time scale, compensated by φi. These fluctuations can explain much of the shift variation.


Assuntos
Imageamento por Ressonância Magnética , Proteínas , Temperatura , Conformação Proteica , Espectroscopia de Ressonância Magnética , Proteínas/química , Ressonância Magnética Nuclear Biomolecular
7.
Chem Commun (Camb) ; 60(22): 3083-3086, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38407363

RESUMO

With perdeuteration, solid-state NMR spectroscopy of large proteins suffers from incomplete amide-proton back-exchange. Using a 72 kDa micro-crystalline protein, we show that deuteration exclusively via deuterated amino acids, well-established in solution to suppress sidechain protonation without proton back-exchange obstacles, provides spectral resolution comparable to perdeuterated preparations at intermediate spinning frequencies.


Assuntos
Aminoácidos , Prótons , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Espectroscopia de Ressonância Magnética
8.
J Am Chem Soc ; 146(8): 5063-5066, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38373110

RESUMO

Protein-ligand interactions can be detected by observing changes in the transverse relaxation rates of the ligand upon binding. The ultrafast NMR technique, which correlates the chemical shift with the transverse relaxation rate, allows for the simultaneous acquisition of R2 for carbon spins at different positions. In combination with dissolution dynamic nuclear polarization (D-DNP), where the signal intensity is enhanced by thousands of times, the R2 values of several carbon signals from unlabeled benzylamine are observable within a single scan. The hyperpolarized ultrafast chemical shift-R2 correlated experiment separates chemical shift encoding from the readout phase in the NMR pulse sequence, which allows it to beat the fundamental limit on the spectral resolution otherwise imposed by the sampling theorem. Applications enabled by the ability to measure multiple relaxation rates in a single scan include the study of structural properties of protein-ligand interactions.


Assuntos
Carbono , Proteínas , Ressonância Magnética Nuclear Biomolecular/métodos , Ligantes , Proteínas/química , Espectroscopia de Ressonância Magnética/métodos
9.
J Phys Chem B ; 128(10): 2293-2303, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38417154

RESUMO

The coexistence of rigid and mobile molecules or molecular segments abounds in biomolecular assemblies. Examples include the carbohydrate-rich cell walls of plants and intrinsically disordered proteins that contain rigid ß-sheet cores. In solid-state nuclear magnetic resonance (NMR) spectroscopy, dipolar polarization transfer experiments are well suited for detecting rigid components, whereas scalar-coupling experiments are well suited for detecting highly mobile components. However, few NMR methods are available to detect the segments that undergo intermediate-amplitude fast motion. Here, we introduce two NMR experiments, a two-dimensional T2H-filtered CP-hCH correlation and a three-dimensional J-INADEQUATE CCH correlation, to observe this intermediate-amplitude motion. Both experiments involve 1H detection under fast magic-angle spinning (MAS). By combining 1H transverse relaxation (T2H) filters with dipolar polarization transfer, we suppress the signals of both highly rigid and highly mobile species, thus revealing the signals of intermediate mobile species. 1H detection under fast MAS is crucial for distinguishing the different motional amplitudes. We demonstrate these techniques on several plant cell wall samples and show that they allow the selective detection and resolution of certain hemicellulose and pectin signals, which are usually masked by the signals of the rigid cellulose and the highly dynamic pectins in purely dipolar and scalar NMR spectra.


Assuntos
Celulose , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos
10.
J Phys Chem Lett ; 15(7): 1930-1935, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38346015

RESUMO

Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (Kd,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (Kd,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The NMR approach developed herein can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies.


Assuntos
Imageamento por Ressonância Magnética , Meliteno , Ressonância Magnética Nuclear Biomolecular/métodos , Modelos Moleculares , Espectroscopia de Ressonância Magnética
11.
J Phys Chem B ; 128(7): 1711-1723, 2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38348474

RESUMO

Polypeptides often self-assemble to form amyloid fibrils, which contain cross-ß structural motifs and are typically 5-15 nm in width and micrometers in length. In many cases, short segments of longer amyloid-forming protein or peptide sequences also form cross-ß assemblies but with distinctive ribbon-like morphologies that are characterized by a well-defined thickness (on the order of 5 nm) in one lateral dimension and a variable width (typically 10-100 nm) in the other. Here, we use a novel combination of data from solid-state nuclear magnetic resonance (ssNMR), dark-field transmission electron microscopy (TEM), atomic force microscopy (AFM), and cryogenic electron microscopy (cryoEM) to investigate the structures within amyloid ribbons formed by residues 14-23 and residues 11-25 of the Alzheimer's disease-associated amyloid-ß peptide (Aß14-23 and Aß11-25). The ssNMR data indicate antiparallel ß-sheets with specific registries of intermolecular hydrogen bonds. Mass-per-area values are derived from dark-field TEM data. The ribbon thickness is determined from AFM images. For Aß14-23 ribbons, averaged cryoEM images show a periodic spacing of ß-sheets. The combined data support structures in which the amyloid ribbon growth direction is the direction of intermolecular hydrogen bonds between ß-strands, the ribbon thickness corresponds to the width of one ß-sheet (i.e., approximately the length of one molecule), and the variable ribbon width is a variable multiple of the thickness of one ß-sheet (i.e., a multiple of the repeat distance in a stack of ß-sheets). This architecture for a cross-ß assembly may generally exist within amyloid ribbons.


Assuntos
Amiloide , Elétrons , Microscopia de Força Atômica , Estrutura Secundária de Proteína , Ressonância Magnética Nuclear Biomolecular/métodos , Amiloide/química , Proteínas Amiloidogênicas , Peptídeos beta-Amiloides/química
12.
Proc Natl Acad Sci U S A ; 121(8): e2301053120, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38346186

RESUMO

While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C'-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra.


Assuntos
Temperatura Baixa , Proteínas , Temperatura , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas/química , Ressonância Magnética Nuclear Biomolecular
13.
J Am Chem Soc ; 146(4): 2319-2324, 2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38251829

RESUMO

Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Here, using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 µM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Conformação Proteica
14.
J Am Chem Soc ; 146(6): 3825-3835, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38293947

RESUMO

Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.


Assuntos
Proteínas de Transporte , Imageamento por Ressonância Magnética , Deutério , Espectroscopia de Ressonância Magnética/métodos , Radical Hidroxila , Polissacarídeos , Ressonância Magnética Nuclear Biomolecular/métodos
15.
J Med Chem ; 67(3): 1701-1733, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38290426

RESUMO

The drug discovery landscape has undergone a significant transformation over the past decade, owing to research endeavors in a wide range of areas leading to strategies for pursuing new drug targets and the emergence of novel drug modalities. NMR spectroscopy has been a technology of fundamental importance to these research pursuits and has seen its use expanded both within and outside of traditional medicinal chemistry applications. In this perspective, we will present advancement of NMR-derived methods that have facilitated the characterization of small molecules and novel drug modalities including macrocyclic peptides, cyclic dinucleotides, and ligands for protein degradation. We will discuss innovations in NMR spectroscopy at the chemistry and biology interface that have broadened NMR's utility from hit identification through lead optimization activities. We will also discuss the promise of emerging NMR approaches in bridging our understanding and addressing challenges in the pursuit of the therapeutic agents of the future.


Assuntos
Descoberta de Drogas , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Descoberta de Drogas/métodos , Ligação Proteica , Química Farmacêutica , Ligantes , Ressonância Magnética Nuclear Biomolecular/métodos
16.
Chemphyschem ; 25(4): e202300565, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38175858

RESUMO

Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid-state NMR to assess rotameric inter-conversions and other dynamic modes of the methionine in the context of a nine-residue random-coil peptide (RC9) with the low-complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine-containing systems, characterized previously. Namely, the M35 of hydrated amyloid-ß1-40 in the three-fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)-methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant.


Assuntos
Peptídeos beta-Amiloides , Metionina , Temperatura , Espectroscopia de Ressonância Magnética , Peptídeos beta-Amiloides/química , Racemetionina , Ressonância Magnética Nuclear Biomolecular
17.
Angew Chem Int Ed Engl ; 63(9): e202316273, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38185473

RESUMO

Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2-19 F, 2-13 C]-adenosine, which allow the incorporation of aromatic 19 F-13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2-19 F, 2-13 C]-adenosine-phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2-19 F, 2-13 C]-adenosine triphosphate, the 124 nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2-19 F, 2-13 C]-A-labeled RNA featured sharper resonances than the [2-1 H, 2-13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2-19 F, 2-13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2-19 F, 2-13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells.


Assuntos
Adenosina , RNA , Humanos , Espectroscopia de Ressonância Magnética/métodos , RNA/química , Nucleotídeos , Trifosfato de Adenosina , Ressonância Magnética Nuclear Biomolecular/métodos
18.
J Mol Biol ; 436(4): 168438, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38185323

RESUMO

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.


Assuntos
Domínio Catalítico , Cisteína , Doenças Neurodegenerativas , Ubiquitina Tiolesterase , Humanos , Sítios de Ligação/genética , Cisteína/química , Cisteína/genética , Cinética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Espalhamento a Baixo Ângulo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/genética , Difração de Raios X , Doenças Neurodegenerativas/genética
19.
Sci Data ; 11(1): 30, 2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38177162

RESUMO

Multidimensional NMR spectra are the basis for studying proteins by NMR spectroscopy and crucial for the development and evaluation of methods for biomolecular NMR data analysis. Nevertheless, in contrast to derived data such as chemical shift assignments in the BMRB and protein structures in the PDB databases, this primary data is in general not publicly archived. To change this unsatisfactory situation, we present a standardized set of solution NMR data comprising 1329 2-4-dimensional NMR spectra and associated reference (chemical shift assignments, structures) and derived (peak lists, restraints for structure calculation, etc.) annotations. With the 100-protein NMR spectra dataset that was originally compiled for the development of the ARTINA deep learning-based spectra analysis method, 100 protein structures can be reproduced from their original experimental data. The 100-protein NMR spectra dataset is expected to help the development of computational methods for NMR spectroscopy, in particular machine learning approaches, and enable consistent and objective comparisons of these methods.


Assuntos
Imageamento por Ressonância Magnética , Proteínas , Algoritmos , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química
20.
J Struct Biol ; 216(1): 108061, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38185342

RESUMO

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.


Assuntos
Durapatita , Vitronectina , Humanos , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular/métodos
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