RESUMO
It is common for NMR relaxation dispersion experiments to suggest an absence of dynamics despite anecdotal indications of conformational dynamics. We explore the potential explanations and approaches to this conundrum. Some inconsistencies have been observed between two relaxation dispersion experiments, Carr-Purcell-Meiboom-Gill (CPMG) and adiabatic relaxation dispersion experiments, in recent dynamic studies of different biomolecules. Theoretical analyses show that such seemingly paradoxical results might come from a complex exchange topology that is concealed by the application of the simple two-site exchange model for interpretation of the relaxation dispersion data. Scenarios are explored and revealed in which the presence of complex millisecond conformational exchange could suppress the amplitude of CPMG relaxation dispersion profiles, even when the exchange rates are within the detectable range of the experiments. With experimental errors, the suppressed relaxation dispersion profiles could lead to the conclusion of "no millisecond conformational exchange". However, such hidden dynamics can potentially be detected by adiabatic relaxation dispersion experiments. Finally, we demonstrate some advantages of adiabatic relaxation dispersion experiments over conventional relaxation dispersion experiments and a simplified computational approach to analyze the adiabatic relaxation dispersion profiles.
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KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-µs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas p21(ras) , Fenômenos Fisiológicos Celulares , Humanos , Conformação Molecular , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas ras/químicaRESUMO
Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods-expressed protein ligation, protein trans-splicing, and native chemical ligation-each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Engenharia de Proteínas/métodos , Staphylococcus aureus/enzimologia , Domínios ProteicosRESUMO
Recent methyl adiabatic relaxation dispersion experiments provide examination of conformational dynamics across a very wide timescale (102-105 s-1) and, particularly, provide insight into the hydrophobic core of proteins and allosteric effects associated with modulators. The experiments require efficient decoupling of 1H and 13C spin interactions, and some artifacts have been discovered, which are associated with the design of the proton decoupling scheme. The experimental data suggest that the original design is valid; however, pulse sequences with either no proton decoupling or proton decoupling with imperfect pulses can potentially exhibit complications in the experiments. Here, we demonstrate that pulse imperfections in the proton decoupling scheme can be dramatically alleviated by using a single composite π pulse and provide pure single-exponential relaxation data. It allows the opportunity to access high-quality methyl adiabatic relaxation dispersion data by removing the cross-correlation between dipole-dipole interaction and chemical shift anisotropy. The resulting high-quality data is illustrated with the binding of an allosteric modulator (G2BR) to the ubiquitin conjugating enzyme Ube2g2.
Assuntos
Artefatos , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Conformação Proteica , Receptores do Fator Autócrino de Motilidade/química , Enzimas de Conjugação de Ubiquitina/química , Regulação Alostérica , Sítios de Ligação , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Dobramento de Proteína , Prótons , Receptores do Fator Autócrino de Motilidade/genética , Proteínas Recombinantes de Fusão/metabolismo , Termodinâmica , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Methyl-TROSY is one of the most powerful NMR spectroscopic tools for studying structures and conformational dynamics of large protein complexes in solution. In studying conformational dynamics, side chains usually display heterogeneous dynamics, including collective and local motions, that can be difficult to detect and analyze by conventional relaxation dispersion (RD) approaches. The combination of NH-based heteronuclear adiabatic relaxation dispersion (HARD) experiments and a geometric approximation (geoHARD) has been shown to have several advantages over conventional RD in revealing conformational dynamics over a broad time scale. Here, we demonstrate a new technique that has been developed to detect both heterogeneous and wide time scale conformational dynamics in the hydrophobic interior of large macromolecules utilizing methyl-geoHARD. It is shown that methyl-geoHARD will be feasible at ultrahigh magnetic fields (>1 GHz), when this technology becomes available. For the ZA domain of Arf-GAP ASAP1, with a global correlational time of 24 ns at 15 °C, a wide range of conformational dynamics (exhibiting chemical exchange rates (kex) between 102 and 105 s-1) are observed in the methyl groups of isoleucine, leucine, and valine. The dynamics include collective and independent local motions. Furthermore, portions of the collective motions have been confirmed by single-quantum Carr-Purcell-Meiboom-Gill (SQ-CPMG) RD experiments; however, motions outside of the detectable CPMG window (400-8000 s-1) cannot be accurately determined by SQ-CPMG experiments. The methyl-geoHARD experiment allows the dissection of heterogeneous conformational dynamics and pinpoints important motions that, potentially, can be correlated with important biological functions and recognition.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/química , Biocatálise , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Teoria Quântica , Fatores de TempoRESUMO
A new computational strategy is reported that provides a fast approximation of numerical solutions of differential equations in general. The method is demonstrated with the analysis of NMR adiabatic relaxation dispersion experiments to reveal biomolecular dynamics. When an analytical solution to the theoretical equations describing a physical process is not available, the new approach can significantly accelerate the computational speed of the conventional numerical integration up to 10(5) times. NMR adiabatic relaxation dispersion experiments enhanced with optimized proton-decoupled pulse sequences, although extremely powerful, have previously been refractory to quantitative analysis. Both simulations and experimental validation demonstrate detectable "slow" (microsecond to millisecond) conformational exchange rates from 10(2) to 10(5) s(-1). This greatly expanded time-scale range enables the characterization of a wide array of conformational fluctuations for individual residues, which correlate with biomolecular function and were previously inaccessible. Moreover, the new computational method can be potentially generalized for analysis of new types of relaxation dispersion experiments to characterize the various dynamics of biomolecular systems.
Assuntos
Biologia Computacional/métodos , Modelos Teóricos , Ressonância Magnética Nuclear Biomolecular/métodos , Enzimas de Conjugação de Ubiquitina/química , Simulação por Computador , Cinética , Conformação Proteica , PrótonsRESUMO
Engineering functional protein scaffolds capable of carrying out chemical catalysis is a major challenge in enzyme design. Starting from a noncatalytic protein scaffold, we recently generated a new RNA ligase by in vitro directed evolution. This artificial enzyme lost its original fold and adopted an entirely new structure with substantially enhanced conformational dynamics, demonstrating that a primordial fold with suitable flexibility is sufficient to carry out enzymatic function.
Assuntos
Catálise , Engenharia de Proteínas/métodos , RNA Ligase (ATP)/química , Alanina/química , Sequência de Aminoácidos , Domínio Catalítico , Evolução Molecular Direcionada/métodos , Enzimas/química , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Metais/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Dobramento de ProteínaRESUMO
The KRAS gene plays a pivotal role in numerous cancers by encoding a GTPase that upon association with the plasma membrane activates the MAPK pathway, promoting cellular proliferation. In our study, we investigated small molecules that disrupt KRAS's membrane interaction, hypothesizing that such disruption could in turn inhibit mutant RAS signaling. Native mass spectrometry screening of KRAS-FMe identified compounds with a preference for interacting with the hypervariable region (HVR), and surface plasmon resonance (SPR) further refined our selection to graveoline as a compound exhibiting preferential HVR binding. Subsequent nuclear magnetic resonance (NMR) analysis showed that graveoline's interaction with KRAS depends on C-terminal O-methylation. Moreover, our findings revealed multiple interaction sites, suggesting weak engagement with the KRAS G domain. Using nanodiscs as a membrane mimetic, further characterization through NMR and Förster resonance energy transfer (FRET) studies demonstrated graveoline's ability to perturb KRAS membrane interaction in a biochemical setting. Our biophysical approach sheds light on the intricate molecular mechanisms underlying KRAS-ligand interactions, providing valuable insights into understanding the KRAS-associated pathophysiology. These findings contribute to the translational aspect of our study, offering potential avenues for further research targeting KRAS membrane association with the potential to lead to a new class of RAS therapeutics.
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Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.
Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Proteínas ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Proteínas ras/metabolismo , Conformação Proteica , Espectroscopia de Ressonância MagnéticaRESUMO
NMR relaxation dispersion experiments have been widely applied to probe important conformational exchange of macro-molecules in many biological systems. The current improvements in computational techniques as well as the theoretical breakthroughs make the quantitative data analysis of complex exchange models possible. However, the topology of a given exchange model is also one of the main factors affecting the solution of Bloch-McConnell equation. The lack of a theoretical analysis of the exchange topologies at n-site exchange hinders further progress of such data analysis. Here, using graph theory, we reveal the topological complexity of n-site exchange and present all exchange models when n is less than 6. Furthermore, we introduce an alternative way, using machine learning, to select an exchange model based on a set of relaxation dispersion data without fitting them with every individual exchange model.
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Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear BiomolecularRESUMO
The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains--the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)--flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the "coupled-allostery" model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.
Assuntos
Proteínas do Nucleocapsídeo/química , Ribonucleoproteínas/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus , Ensaio de Desvio de Mobilidade Eletroforética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Ligação Proteica , Estrutura Secundária de Proteína , RNA Viral/metabolismo , Ribonucleoproteínas/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
Structural biology often focuses primarily on three-dimensional structures of biological macromolecules, deposited in the Protein Data Bank (PDB). This resource is a remarkable entity for the world-wide scientific and medical communities, as well as the general public, as it is a growing translation into three-dimensional space of the vast information in genomic databases, e.g. GENBANK. There is, however, significantly more to understanding biological function than the three-dimensional coordinate space for ground-state structures of biomolecules. The vast array of biomolecules experiences natural dynamics, interconversion between multiple conformational states, and molecular recognition and allosteric events that play out on timescales ranging from picoseconds to seconds. This wide range of timescales demands ingenious and sophisticated experimental tools to sample and interpret these motions, thus enabling clearer insight into functional annotation of the PDB. NMR spectroscopy is unique in its ability to sample this range of timescales at atomic resolution and in physiologically relevant conditions using spin relaxation methods. The field is constantly expanding to provide new creative experiments, to yield more detailed coverage of timescales, and to broaden the power of interpretation and analysis methods. This review highlights the current state of the methodology and examines the extension of analysis tools for more complex experiments and dynamic models. The future for understanding protein dynamics is bright, and these extended tools bring greater compatibility with developments in computational molecular dynamics, all of which will further our understanding of biological molecular functions. These facets place NMR as a key component in integrated structural biology.
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Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Humanos , Conformação ProteicaRESUMO
The Carr-Purcell-Meiboom-Gill (CPMG) experiment is one of the most classical and well-known relaxation dispersion experiments in NMR spectroscopy, and it has been successfully applied to characterize biologically relevant conformational dynamics in many cases. Although the data analysis of the CPMG experiment for the 2-site exchange model can be facilitated by analytical solutions, the data analysis in a more complex exchange model generally requires computationally-intensive numerical analysis. Recently, a powerful computational strategy, geometric approximation, has been proposed to provide approximate numerical solutions for the adiabatic relaxation dispersion experiments where analytical solutions are neither available nor feasible. Here, we demonstrate the general potential of geometric approximation by providing a data analysis solution of the CPMG experiment for both the traditional 2-site model and a linear 3-site exchange model. The approximate numerical solution deviates less than 0.5% from the numerical solution on average, and the new approach is computationally 60,000-fold more efficient than the numerical approach. Moreover, we find that accurate dynamic parameters can be determined in most cases, and, for a range of experimental conditions, the relaxation can be assumed to follow mono-exponential decay. The method is general and applicable to any CPMG RD experiment (e.g. N, C', Cα, Hα, etc.) The approach forms a foundation of building solution surfaces to analyze the CPMG experiment for different models of 3-site exchange. Thus, the geometric approximation is a general strategy to analyze relaxation dispersion data in any system (biological or chemical) if the appropriate library can be built in a physically meaningful domain.
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Espectroscopia de Ressonância Magnética/métodos , Algoritmos , Simulação por Computador , Interpretação Estatística de Dados , Conformação ProteicaRESUMO
Eukaryotic protein kinases (EPKs) constitute a class of allosteric switches that mediate a myriad of signaling events. It has been postulated that EPKs' active and inactive states depend on the structural architecture of their hydrophobic cores, organized around two highly conserved spines: C-spine and R-spine. How the spines orchestrate the transition of the enzyme between catalytically uncommitted and committed states remains elusive. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we found that the hydrophobic core of the catalytic subunit of protein kinase A, a prototypical and ubiquitous EPK, moves synchronously to poise the C subunit for catalysis in response to binding adenosine 5'-triphosphate. In addition to completing the C-spine, the adenine ring fuses the ß structures of the N-lobe and the C-lobe. Additional residues that bridge the two spines (I150 and V104) are revealed as part of the correlated hydrophobic network; their importance was validated by mutagenesis, which led to inactivation. Because the hydrophobic architecture of the catalytic core is conserved throughout the EPK superfamily, the present study suggests a universal mechanism for dynamically driven allosteric activation of kinases mediated by coordinated signal transmission through ordered motifs in their hydrophobic cores.
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Trifosfato de Adenosina/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Modelos Moleculares , Regulação Alostérica , Domínio Catalítico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear BiomolecularRESUMO
We present an enhanced version of the FLAMEnGO (Fuzzy Logic Assignment of Methyl Group) software, a structure-based method to assign methyl group resonances in large proteins. FLAMEnGO utilizes a fuzzy logic algorithm coupled with Monte Carlo sampling to obtain a probability-based assignment of the methyl group resonances. As an input, FLAMEnGO requires either the protein X-ray structure or an NMR structural ensemble including data such as methyl-methyl NOESY, paramagnetic relaxation enhancement (PRE), methine-methyl TOCSY data. Version 2.0 of this software (FLAMEnGO 2.0) has a user-friendly graphic interface and presents improved modules that enable the input of partial assignments and additional NMR restraints. We tested the performance of FLAMEnGO 2.0 on maltose binding protein (MBP) as well as the C-subunit of the cAMP-dependent protein kinase A (PKA-C). FLAMEnGO 2.0 can be used as a standalone method or to assist in the completion of partial resonance assignments and can be downloaded at www.chem.umn.edu/groups/veglia/forms/flamengo2-form.html.
Assuntos
Algoritmos , Proteínas Quinases Dependentes de AMP Cíclico/química , Lógica Fuzzy , Proteínas Ligantes de Maltose/química , Metano/análogos & derivados , Ressonância Magnética Nuclear Biomolecular/métodos , Software , Metano/análise , Metano/química , Método de Monte Carlo , Reconhecimento Automatizado de Padrão , Interface Usuário-ComputadorRESUMO
Building on a recent method by Matthews and co-workers [1], we developed a new and efficient algorithm to assign methyl resonances from sparse and ambiguous NMR data. The new algorithm (FLAMEnGO: Fuzzy Logic Assignment of MEthyl GrOups) uses Monte Carlo sampling in conjunction with fuzzy logic to obtain the assignment of methyl resonances at high fidelity. Furthermore, we demonstrate that the inclusion of paramagnetic relaxation enhancement (PRE) data in the assignment strategy increases the percentage of correct assignments with sparse NOE data. Using synthetic tests and experimental data we show that this new approach provides up to â¼80% correct assignments with only 30% of methyl-methyl NOE data. In the experimental case of ubiquitin, PRE data from two spin labeled sites improve the percentage of assigned methyl groups up to â¼91%. This new strategy promises to further expand methyl group NMR spectroscopy to very large macromolecular systems.
Assuntos
Algoritmos , Lógica Fuzzy , Espectroscopia de Ressonância Magnética/métodos , Metano/análogos & derivados , Ubiquitina/análise , Ubiquitina/química , Metano/análise , Metano/química , Reconhecimento Automatizado de Padrão/métodosRESUMO
NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R(1ρ) and Carr-Purcell-Meiboom-Gill (CPMG) R(2) experiments are commonly used to characterize µs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R(1ρ) and transverse R(2ρ)) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (k(ex)â¼10(4)-10(5) s(-1)). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R(1ρ) and R(2ρ) relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R(1ρ) and R(2ρ) that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R(1ρ) experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent.