RESUMO
A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH2 groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing 15N and 1H components of NH2 multiplets. Although even modest sensitivity gains in 2D NH2-TROSY correlation maps compared to their decoupled NH2-HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH2 sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH2-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH2 positions of the L99A T4 lysozyme mutant - a model system for studies of the role of protein dynamics in ligand binding.
RESUMO
Proline isomerization is widely recognized as a kinetic bottleneck in protein folding, amplified for proteins rich in Pro residues. We introduced repeated hydrostatic pressure jumps between native and pressure-denaturing conditions inside an NMR sample cell to study proline isomerization in the pressure-sensitized L50A ubiquitin mutant. Whereas in two unfolded heptapeptides, X-Pro peptide bonds isomerized ca 1.6-fold faster at 1 bar than at 2.5 kbar, for ubiquitin ca eight-fold faster isomerization was observed for Pro-38 and ca two-fold for Pro-19 and Pro-37 relative to rates measured in the pressure-denatured state. Activation energies for isomerization in pressure-denatured ubiquitin were close to literature values of 20 kcal/mole for denatured polypeptides but showed a substantial drop to 12.7 kcal/mole for Pro-38 at atmospheric pressure. For ubiquitin isomers with a cis E18-P19 peptide bond, the 1-bar NMR spectrum showed sharp resonances with near random coil chemical shifts for the C-terminal half of the protein, characteristic of an unfolded chain, while most of the N-terminal residues were invisible due to exchange broadening, pointing to a metastable partially folded state for this previously recognized 'folding nucleus'. For cis-P37 isomers, a drop in pressure resulted in the rapid loss of nearly all unfolded-state NMR resonances, while the recovery of native state intensity revealed a slow component attributed to cis â trans isomerization of P37. This result implies that the NMR-invisible cis-P37 isomer adopts a molten globule state that encompasses the entire length of the ubiquitin chain, suggestive of a structure that mostly resembles the folded state.
Assuntos
Peptídeos , Prolina , Desnaturação Proteica , Dobramento de Proteína , Ubiquitina , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Pressão , Prolina/química , Conformação Proteica , Ubiquitina/química , Peptídeos/químicaRESUMO
Amelotin is an intrinsically disordered protein (IDP) rich in Pro residues and is involved in hydroxyapatite mineralization. It rapidly oligomerizes under physiological conditions of pH and pressure but reverts to its monomeric IDP state at elevated pressure. We identified a 105-residue segment of the protein that becomes ordered upon oligomerization, and we used pressure-jump NMR spectroscopy to measure long-range NOE contacts that exist exclusively in the oligomeric NMR-invisible state. The kinetics of oligomerization and dissociation were probed at the residue-specific level, revealing that the oligomerization process is initiated in the C-terminal half of the segment. Using pressure-jump NMR, the degree of order in the oligomer at the sites of Pro residues was probed by monitoring changes in cis/trans equilibria relative to the IDP state after long-term equilibration under oligomerizing conditions. Whereas most Pro residues revert to trans in the oligomeric state, Pro-49 favors a cis configuration and three Pro residues retain an unchanged cis fraction, pointing to their local lack of order in the oligomeric state. NOE contacts and secondary 13C chemical shifts in the oligomeric state indicate the presence of an 11-residue α-helix, preceded by a small intramolecular antiparallel ß-sheet, with slower formation of long-range intermolecular interactions to N-terminal residues. Although none of the models generated by AlphaFold2 for the amelotin monomer was consistent with experimental data, subunits of a hexamer generated by AlphaFold-Multimer satisfied intramolecular NOE and chemical shift data and may provide a starting point for developing atomic models for the oligomeric state.
Assuntos
Prolina , Proteínas , Conformação Proteica , Isomerismo , Prolina/química , Espectroscopia de Ressonância Magnética/métodosRESUMO
The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro /Nsp5), is a key antiviral drug target. NMR spectroscopy of this large system proved challenging and resonance assignments have remained incomplete. Here we present the near-complete (>97 %) backbone assignments of a C145A variant of Mpro (Mpro C145A ) both with, and without, the N-terminal auto-cleavage substrate sequence, in its native homodimeric state. We also present SILLY (Selective Inversion of thioL and Ligand for NOESY), a simple yet effective pseudo-3D NMR experiment that utilizes NOEs to identify interactions between Cys-thiol or aliphatic protons, and their spatially proximate backbone amides in a perdeuterated protein background. High protection against hydrogen exchange is observed for 10 of the 11 thiol groups in Mpro C145A , even those that are partially accessible to solvent. A combination of SILLY methods and high-resolution triple-resonance NMR experiments reveals site-specific interactions between Mpro , its substrate peptides, and other ligands, which present opportunities for competitive binding studies in future drug design efforts.
Assuntos
COVID-19 , Prótons , Amidas , Antivirais/química , Proteases 3C de Coronavírus , Cisteína Endopeptidases/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Peptídeos/metabolismo , Inibidores de Proteases , SARS-CoV-2 , Solventes , Compostos de SulfidrilaRESUMO
The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N-1HN and 13C'-1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.
Assuntos
Proteases 3C de Coronavírus/química , Cristalografia por Raios X/métodos , SARS-CoV-2 , COVID-19 , Domínio Catalítico , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Dinâmica Molecular , Conformação Proteica , Dobramento de Proteína , Software , Raios XRESUMO
Chemical denaturation is a well-established approach for probing the equilibrium between folded and unfolded states of proteins. We demonstrate applicability of this method to the detection of a small population of a transiently folded structural element in a system that is often considered to be intrinsically fully disordered. The 1HN, 15N, 13Cα, and 13C' chemical shifts of Aß1-40 and Aß1-42 peptides and their M35-oxidized variants were monitored as a function of urea concentration and compared to analogous urea titrations of synthetic pentapeptides of homologous sequence. Fitting of the chemical shift titrations yields a 10 ± 1% population for a structured element at the C-terminus of Aß1-42 that folds with a cooperativity of m = 0.06 kcal/mol·M. The fit also yields the chemical shifts of the folded state and, using a database search, for Aß1-42 these shifts identified an antiparallel intramolecular ß-sheet for residues I32-A42, linked by a type I' ß-turn at G37 and G38. The structure is destabilized by oxidation of M35. Paramagnetic relaxation rates and two previously reported weak, medium-range NOE interactions are consistent with this transient ß-sheet. Introduction of the requisite A42C mutation and tagging with MTSL resulted in a small stabilization of this ß-sheet. Chemical shift analysis suggests a C-terminal ß-sheet may be present in Aß1-40 too, but the turn type at G37 is not type I'. The approach to derive Transient Structure from chemical Denaturation by NMR (TSD-NMR), demonstrated here for Aß peptides, provides a sensitive tool for identifying the presence of lowly populated, transiently ordered elements in proteins that are considered to be intrinsically disordered, and permits extraction of structural data for such elements.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Desnaturação Proteica , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em Folha beta , Dobramento de ProteínaRESUMO
Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and 1H back-exchange of the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2â¯×â¯306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the 1H-1H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonance assignments as well as for probing structural features.
RESUMO
Although the concepts of nonuniform sampling (NUSâââââââ) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago , it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection of closely spaced peaks (i.e., "resolution") or peaks of weak intensity (i.e., "sensitivity"). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the "Nonuniform Sampling Contest" (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform.
RESUMO
Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. While the impact of pressure on the viscosity of water is well known, and our water diffusivity measurements agree closely with theoretical expectations, we find that elevated pressures increase the Rh of dioxane and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction indicated by 13C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurement of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure or urea concentration shows that Rh values of both the folded and the unfolded states remain nearly invariant. At ca 23 Å, the Rh of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) α-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of unfolded ubiquitin and α-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Sinucleínas/química , Ubiquitina/química , Ureia/química , Difusão , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates the exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated the effect, using NMR spectroscopy, of substituting key charged Arg, Lys, Glu, and Asp residues by Gly or His. Results indicate that such mutations result in the transient breaking of the helix at the site of mutation but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH stability. Whereas a single Gly substitution caused transient breaks â¼20% of the time, two consecutive Gly substitutions break the helix â¼65% of the time. NMR relaxation measurements indicate that the exchange rate between an intact and a broken helix is fast (>300,000 s-1) and that for the wild-type sequence, the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, in which the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines.
Assuntos
Cadeias Pesadas de Miosina , Espectroscopia de Ressonância Magnética , Conformação Proteica em alfa-HéliceRESUMO
Small heat-shock proteins (sHSPs) are molecular chaperones that respond to cellular stresses to combat protein aggregation. HSP27 is a critical human sHSP that forms large, dynamic oligomers whose quaternary structures and chaperone activities depend on environmental factors. Upon exposure to cellular stresses, such as heat shock or acidosis, HSP27 oligomers can dissociate into dimers and monomers, which leads to significantly enhanced chaperone activity. The structured core of the protein, the α-crystallin domain (ACD), forms dimers and can prevent the aggregation of substrate proteins to a similar degree as the full-length protein. When the ACD dimer dissociates into monomers, it partially unfolds and exhibits enhanced activity. Here, we used solution-state NMR spectroscopy to characterize the structure and dynamics of the HSP27 ACD monomer. Web show that the monomer is stabilized at low pH and that its backbone chemical shifts, 15N relaxation rates, and 1H-15N residual dipolar couplings suggest structural changes and rapid motions in the region responsible for dimerization. By analyzing the solvent accessible and buried surface areas of sHSP structures in the context of a database of dimers that are known to dissociate into disordered monomers, we predict that ACD dimers from sHSPs across all kingdoms of life may partially unfold upon dissociation. We propose a general model in which conditional disorder-the partial unfolding of ACDs upon monomerization-is a common mechanism for sHSP activity.
Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Desdobramento de ProteínaRESUMO
Although the order of the time steps in which the non-uniform sampling (NUS) schedule is implemented when acquiring multi-dimensional NMR spectra is of limited importance when sample conditions remain unchanged over the course of the experiment, it is shown to have major impact when samples are unstable. In the latter case, time-ordering of the NUS data points by the normalized radial length yields a reduction of sampling artifacts, regardless of the spectral reconstruction algorithm. The disadvantage of time-ordered NUS sampling is that halting the experiment prior to its completion will result in lower spectral resolution, rather than a sparser data matrix. Alternatively, digitally correcting for sample decay prior to reconstruction of randomly ordered NUS data points can mitigate reconstruction artifacts, at the cost of somewhat lower sensitivity. Application of these sampling schemes to the Alzheimer's amyloid beta (Aß1-42) peptide at an elevated concentration, low temperature, and 3 kbar of pressure, where approximately 75% of the peptide reverts to an NMR-invisible state during the collection of a 3D 15N-separated NOESY spectrum, highlights the improvement in artifact suppression and reveals weak medium-range NOE contacts in several regions, including the C-terminal region of the peptide.
Assuntos
Peptídeos beta-Amiloides/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Manejo de Espécimes/métodos , Algoritmos , Artefatos , TempoRESUMO
Although the α-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a protein's structure. Highly charged single α-helical (SAH) domains, consisting of a high percentage (>75%) of Arg, Lys, and Glu residues, are exceptions to this rule but have been difficult to characterize structurally. Our study focuses on the 68-residue medial tail domain of myosin-VI, which is found to contain a highly ordered α-helical structure extending from Glu-6 to Lys-63. High hydrogen exchange protection factors (15-150), small (ca. 4 Hz) 3 JHNHα couplings, and a near-perfect fit to an ideal model α-helix for its residual dipolar couplings (RDCs), measured in a filamentous phage medium, support the high regularity of this helix. Remarkably, the hydrogen exchange rates are far more homogeneous than the protection factors derived from them, suggesting that for these transiently broken helices the intrinsic exchange rates derived from the amino acid sequence are not appropriate reference values. 15N relaxation data indicate a very high degree of rotational diffusion anisotropy ( Dâ¥/ D⥠≈ 7.6), consistent with the hydrodynamic behavior predicted for such a long, nearly straight α-helix. Alignment of the helix by a paramagnetic lanthanide ion attached to its N-terminal region shows a decrease in alignment as the distance from the tagging site increases. This decrease yields a precise measure for the persistence length of 224 ± 10 Å at 20 °C, supporting the idea that the role of the SAH helix is to act as an extension of the myosin-VI lever arm.
Assuntos
Cadeias Pesadas de Miosina/química , Domínios Proteicos , Sequência de Aminoácidos , Animais , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , SuínosRESUMO
The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of ß-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mammalian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.
Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Agregação Patológica de Proteínas/genética , Multimerização Proteica/genética , Desdobramento de Proteína , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Chaperonas Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Conformação Proteica em Folha beta/genética , Estrutura Quaternária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Disulfide bridges in proteins are formed by the oxidation of pairs of cysteine residues. These cross-links play a critical role in stabilizing the 3D-structure of small disulfide rich polypeptides such as hormones and venom toxins. The arrangement of the multiple disulfide bonds directs the peptide fold into distinct structural motifs that have evolved for resistance against biochemical and physical insults. These structural scaffolds have, therefore, proven to be very attractive in bioengineering efforts to develop novel biologics with applications in health and agriculture. Structural characterization of small disulfide rich peptides (DRPs) presents unique challenges when using commonly applied biophysical methods. NMR is the most commonly used method for studying such molecules, where the relatively small size of these molecules results in highly precise structural ensembles defined by a large number of distance and dihedral angle restraints per amino acid. However, in NMR the sulfur atoms that are involved in three of the five dihedral angles in a disulfide bond cannot be readily measured. Given the central role of disulfide bonds in the structure of these molecules, it is unclear what the inherent resolution of such NMR structures is when using traditional NMR methods. Here, we use an extensive set of long-range residual dipolar couplings (RDCs) to assess the resolution of the NMR structure of a disulfide-rich peptide. We find that structures based primarily on NOEs, yield ensembles that are equivalent to a crystallographic resolution of 2-3 Å in resolution, and that incorporation of RDCs reduces this to ~1-1.5 Å resolution. At this resolution the sidechain of ordered amino acids can be defined accurately, allowing the geometry of the cysteine bridges to be better defined, and allowing for disulfide-bond connectivities to be determined with high confidence. The observed improvements in resolution when using RDCs is remarkable considering the small size of these peptides.
RESUMO
In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a â¼100-µs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above â¼300 µM, oligomeric off-pathway intermediates compete with folding of the native state.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Ubiquitina/química , Humanos , Pressão HidrostáticaRESUMO
In unfolded proteins, peptide bonds involving Pro residues exist in equilibrium between the minor cis and major trans conformations. Folded proteins predominantly contain trans-Pro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be a rate-limiting step in protein folding. Moreover, kinases and phosphatases that act upon Ser/Thr-Pro motifs exhibit preferential recognition of either the cis- or trans-Pro conformer. Here, NMR spectra obtained at both atmospheric and high pressures indicate that the population of cis-Pro falls well below previous estimates, an effect attributed to the use of short peptides with charged termini in most prior model studies. For the intrinsically disordered protein α-synuclein, cis-Pro populations at all of its five X-Pro bonds are less than 5 %, with only modest ionic strength dependence and no detectable effect of the previously demonstrated interaction between the N- and C-terminal halves of the protein. Comparison to small peptides with the same amino-acid sequence indicates that peptides, particularly those with unblocked, oppositely charged amino and carboxyl end groups, strongly overestimate the amount of cis-Pro.
Assuntos
Prolina/química , alfa-Sinucleína/metabolismo , Isomerismo , Ressonância Magnética Nuclear Biomolecular , Pressão , Desnaturação Proteica , alfa-Sinucleína/químicaRESUMO
The molecular mechanism through which the interaction of a clonotypic αß T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR ß-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cß H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays.
Assuntos
Sítio Alostérico/imunologia , Regiões Determinantes de Complementaridade/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Regiões Determinantes de Complementaridade/metabolismo , Cristalografia por Raios X , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Simulação de Dinâmica Molecular , Mutagênese , Peptídeos/metabolismo , Ligação Proteica/imunologia , Domínios Proteicos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/metabolismoRESUMO
Implementation of a new algorithm, SMILE, is described for reconstruction of non-uniformly sampled two-, three- and four-dimensional NMR data, which takes advantage of the known phases of the NMR spectrum and the exponential decay of underlying time domain signals. The method is very robust with respect to the chosen sampling protocol and, in its default mode, also extends the truncated time domain signals by a modest amount of non-sampled zeros. SMILE can likewise be used to extend conventional uniformly sampled data, as an effective multidimensional alternative to linear prediction. The program is provided as a plug-in to the widely used NMRPipe software suite, and can be used with default parameters for mainstream application, or with user control over the iterative process to possibly further improve reconstruction quality and to lower the demand on computational resources. For large data sets, the method is robust and demonstrated for sparsities down to ca 1%, and final all-real spectral sizes as large as 300 Gb. Comparison between fully sampled, conventionally processed spectra and randomly selected NUS subsets of this data shows that the reconstruction quality approaches the theoretical limit in terms of peak position fidelity and intensity. SMILE essentially removes the noise-like appearance associated with the point-spread function of signals that are a default of five-fold above the noise level, but impacts the actual thermal noise in the NMR spectra only minimally. Therefore, the appearance and interpretation of SMILE-reconstructed spectra is very similar to that of fully sampled spectra generated by Fourier transformation.
Assuntos
Algoritmos , Ressonância Magnética Nuclear Biomolecular/métodos , Simulação por Computador , Análise de Fourier , Sensibilidade e Especificidade , Razão Sinal-Ruído , Software , TempoRESUMO
The interaction between α-synuclein (αS) protein and lipid membranes is key to its role in synaptic vesicle homeostasis and plays a role in initiating fibril formation, which is implicated in Parkinson's disease. The natural state of αS inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence of a tetrameric arrangement, which was reported to be rich in α-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein's Lys C(ε) amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high-resolution homonuclear decoupled nuclear magnetic resonance experiments to probe the reactivity of the 15 αS Lys residues toward N-succinimidyl acetate, effectively half the DSG cross-linker, which results in acetylation of Lys. The intensities of both side chain and backbone amide signals of acetylated Lys residues provide direct information about the reactivity, showing a difference of a factor of 2.5 between the most reactive (K6) and the least reactive (K102) residue. The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease in Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar αS CD signatures. Our data provide new insight into the αS-bilayer interaction, including the pivotal state in which the available lipid surface is limited. Protection of Lys C(ε) amino groups by αS-bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments.