RESUMEN
Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein's conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.
Asunto(s)
Aminoacil-ARNt Sintetasas , Escherichia coli , Simulación de Dinámica Molecular , Polietilenglicoles , Conformación Proteica , Polietilenglicoles/química , Escherichia coli/enzimología , Escherichia coli/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Microscopía de Fuerza Atómica , Dominio Catalítico , Peso MolecularRESUMEN
The effect of molecular crowding on the structure and function of Escherichia coli prolyl-transfer RNA synthetase (Ec ProRS), a member of the aminoacyl-transfer RNA synthetase family, has been investigated using a combined experimental and theoretical method. Ec ProRS is a multidomain enzyme; coupled-domain dynamics are essential for efficient catalysis. To gain insight into the mechanistic detail of the crowding effect, kinetic studies were conducted with varying concentrations and sizes of crowders. In parallel, spectroscopic and quantum chemical studies were employed to probe the "soft interactions" between crowders and protein side chains. Finally, the dynamics of the dimeric protein was examined in the presence of crowders using a long-duration (70 ns) classical molecular dynamic simulations. The results of the simulations revealed a shift in the conformational ensemble, which is consistent with the preferential exclusion of cosolutes. The "soft interactions" model of the crowding effect also explained the alteration in kinetic parameters. In summary, the study found that the effects of molecular crowding on both conformational dynamics and catalytic function are correlated in the multidomain Ec ProRS, an enzyme that is central to protein synthesis in all living cells. This study affirmed that large and small cosolutes have considerable impacts on the structure, dynamics, and function of modular proteins and therefore must be considered for stabilizing protein-based pharmaceuticals and industrial enzymes.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/enzimología , Simulación de Dinámica Molecular , Biocatálisis , Cinética , Dominios Proteicos , TermodinámicaRESUMEN
To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNA(Pro). This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2'-OH of misacylated tRNA(Pro) and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNA(Pro) deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNA(Pro) hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNA(Pro) and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNA(Pro) hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Lisina/genética , Aminoacil-ARNt Sintetasas/química , Dominio Catalítico , Simulación por Computador , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Estructura Terciaria de Proteína , Edición de ARN , ARN de Transferencia de Prolina/metabolismo , TermodinámicaRESUMEN
A literature review is an important part of conducting academic research. Knowing how to conduct a literature search and write a high-quality literature review is a valuable skill. Herein, the authors describe the method of introducing a literature review writing exercise in an upper-level biochemistry course. Since 2020, authors have collaborated with numerous undergraduates writing literature reviews on topics in biochemistry that resulted in peer-reviewed publications. Authors believe that this unique idea of providing a course-based undergraduate research experience (CURE) to many undergraduates, especially those who otherwise do not receive collaborative research experience through traditional research paths, must be shared with other instructors.
Asunto(s)
Bioquímica , Curriculum , Estudiantes , Escritura , Bioquímica/educación , Humanos , Universidades , Literatura de Revisión como AsuntoRESUMEN
Aminoacyl-tRNA synthetases are multidomain enzymes that catalyze covalent attachment of amino acids to their cognate tRNA. Cross-talk between functional domains is a prerequisite for this process. In this study, we investigate the molecular mechanism of site-to-site communication in Escherichia coli prolyl-tRNA synthetase (Ec ProRS). Earlier studies have demonstrated that evolutionarily conserved and/or co-evolved residues that are engaged in correlated motion are critical for the propagation of functional conformational changes from one site to another in modular proteins. Here, molecular simulation and bioinformatics-based analysis were performed to identify dynamically coupled and evolutionarily constrained residues that form contiguous pathways of residue-residue interactions between the aminoacylation and editing domains of Ec ProRS. The results of this study suggest that multiple pathways exist between these two domains to maintain the dynamic coupling essential for enzyme function. Moreover, residues in these interaction networks are generally highly conserved. Site-directed changes of on-pathway residues have a significant impact on enzyme function and dynamics, suggesting that any perturbation along these pathways disrupts the native residue-residue interactions that are required for effective communication between the two functional domains. Free energy analysis revealed that communication between residues within a pathway and cross-talk between pathways are important for coordinating functions of different domains of Ec ProRS for efficient catalysis.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Dominio Catalítico , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Transducción de Señal/fisiología , Aminoacil-ARNt Sintetasas/fisiología , Proteínas de Escherichia coli/fisiología , Simulación de Dinámica MolecularRESUMEN
Polyethylene glycol (PEG) is a polyether compound commonly used in biological research and medicine because it is biologically inert. This simple polymer exists in variable chain lengths (and molecular weights). As they are devoid of any contiguous π-system, PEGs are expected to lack fluorescence properties. However, recent studies suggested the occurrence of fluorescence properties in non-traditional fluorophores like PEGs. Herein, a thorough investigation has been conducted to explore if PEG 20k fluoresces. Results of this combined experimental and computational study suggested that although PEG 20k could exhibit "through-space" delocalization of lone pairs of electrons in aggregates/clusters, formed via intermolecular and intramolecular interactions, the actual contributor of fluorescence between 300 and 400 nm is the stabilizer molecule, i.e., 3-tert-butyl-4-hydroxyanisole present in the commercially available PEG 20k. Therefore, the reported fluorescence properties of PEG should be taken with a grain of salt, warranting further investigation.
RESUMEN
Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern-Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn-Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.
RESUMEN
Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Dominio Catalítico , ADN Bacteriano/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Prolina/metabolismo , Estructura Terciaria de Proteína , ARN de Transferencia de Prolina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de SecuenciaRESUMEN
The redox-dependent changes on the binding between the receptor-binding domain of the severe acute respiratory syndrome-coronavirus-2 spike protein and the peptidase domain of the human cell surface receptor angiotensin-converting enzyme II were investigated by performing molecular dynamics simulations. The reduced states of the protein partners were generated in silico by converting the disulfides to thiols. The role of redox transformation on the protein-protein binding affinity was assessed from the time-evolved structures after 200 ns simulations using electrostatic field calculations and implicit solvation. The present simulations revealed that the bending motion at the protein-protein interface is significantly altered when the disulfides are reduced to thiols. In the native complex, the presence of disulfide bonds preserves the structural complementarity of the protein partners and maintains the intrinsic conformational dynamics. Also, the study demonstrates that when already bound, the disulfide-to-thiol conversion of the receptor-binding domain has a limited impact on the binding of the spike protein to the receptor. However, if the reduction occurs before binding to the receptor, a spectacular conformational change of the receptor-binding domain occurs that fully impairs the binding. In other words, the formation of disulfide bonds, prevalent during oxidative stress, creates a conformation ready to bind to the receptor. Taken together, the present study demonstrates the role of pre-existing oxidative stress in elevating the binding affinity of the spike protein for the human receptor, offering future clues for alternate therapeutic possibilities.
RESUMEN
Using molecular dynamics simulations, the protein-protein interactions of the receptor-binding domain of the wild-type and seven variants of the severe acute respiratory syndrome coronavirus 2 spike protein and the peptidase domain of human angiotensin-converting enzyme 2 were investigated. These variants are alpha, beta, gamma, delta, eta, kappa, and omicron. Using 100 ns simulation data, the residue interaction networks at the protein-protein interface were identified. Also, the impact of mutations on essential protein dynamics, backbone flexibility, and interaction energy of the simulated protein-protein complexes were studied. The protein-protein interface for the wild-type, delta, and omicron variants contained several stronger interactions, while the alpha, beta, gamma, eta, and kappa variants exhibited an opposite scenario as evident from the analysis of the inter-residue interaction distances and pair-wise interaction energies. The study reveals that two distinct residue networks at the central and right contact regions forge stronger binding affinity between the protein partners. The study provides a molecular-level insight into how enhanced transmissibility and infectivity by delta and omicron variants are most likely tied to a handful of interacting residues at the binding interface, which could potentially be utilized for future antibody constructs and structure-based antiviral drug design.
Asunto(s)
Evolución Molecular , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/química , Humanos , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a pathogenic coronavirus causing COVID-19 infection. The interaction between the SARS-CoV-2 spike protein and the human receptor angiotensin-converting enzyme 2, both of which contain several cysteine residues, is impacted by the disulfide-thiol balance in the host cell. The host cell redox status is affected by oxidative stress due to the imbalance between the reactive oxygen/nitrogen species and antioxidants. Recent studies have shown that Vitamin D supplementation could reduce oxidative stress. It has also been proposed that vitamin D at physiological concentration has preventive effects on many viral infections, including COVID-19. However, the molecular-level picture of the interplay of vitamin D deficiency, oxidative stress, and the severity of COVID-19 has remained unclear. Herein, we present a thorough review focusing on the possible molecular mechanism by which vitamin D could alter host cell redox status and block viral entry, thereby preventing COVID-19 infection or reducing the severity of the disease.
Asunto(s)
COVID-19 , Estrés Oxidativo/efectos de los fármacos , SARS-CoV-2/metabolismo , Índice de Severidad de la Enfermedad , Internalización del Virus/efectos de los fármacos , Vitamina D/uso terapéutico , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/patología , COVID-19/prevención & control , Humanos , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an ongoing pandemic of coronavirus disease (COVID-19), which started in 2019. This is a member of Coronaviridae family in the genus Betacoronavirus, which also includes SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). The angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV and SARS-CoV-2 to enter the host cells. In particular, the interaction of viral spike proteins with ACE2 is a critical step in the viral replication cycle. The receptor-binding domain of the viral spike proteins and ACE2 have several cysteine residues. In this study, the role of thiol-disulfide balance on the interactions between SARS-CoV/CoV-2 spike proteins and ACE2 was investigated using molecular dynamics simulations. The study revealed that the binding affinity was significantly impaired when all of the disulfide bonds of both ACE2 and SARS-CoV/CoV-2 spike proteins were reduced to thiol groups. The impact on the binding affinity was less severe when the disulfide bridges of only one of the binding partners were reduced to thiols. This computational finding possibly provides a molecular basis for the differential COVID-19 cellular recognition due to the oxidative stress.
RESUMEN
Novel coronavirus disease 2019 (COVID-19) has resulted in a global pandemic and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several studies have suggested that a precise disulfide-thiol balance is crucial for viral entry and fusion into the host cell and that oxidative stress generated from free radicals can affect this balance. Here, we reviewed the current knowledge about the role of oxidative stress on SARS-CoV and SARS-CoV-2 infections. We focused on the impact of antioxidants, like NADPH and glutathione, and redox proteins, such as thioredoxin and protein disulfide isomerase, that maintain the disulfide-thiol balance in the cell. The possible influence of these biomolecules on the binding of viral protein with the host cell angiotensin-converting enzyme II receptor protein as well as on the severity of COVID-19 infection was discussed.
Asunto(s)
COVID-19/metabolismo , Estrés Oxidativo , SARS-CoV-2/fisiología , Síndrome Respiratorio Agudo Grave/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiología , Acetilcisteína/farmacología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Antivirales/farmacología , Descubrimiento de Drogas , Humanos , Modelos Moleculares , Estrés Oxidativo/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Síndrome Respiratorio Agudo Grave/tratamiento farmacológico , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate tRNAs, an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal, highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic Pro-AMP formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations, as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.
RESUMEN
Enzymes play important roles in many biological processes. Amino acid residues in the active site pocket of an enzyme, which are in direct contact with the substrate(s), are generally believed to be critical for substrate recognition and catalysis. Identifying and understanding how these "catalytic" residues help enzymes achieve enormous rate enhancement has been the focus of many structural and biochemical studies over the past several decades. Recent studies have shown that enzymes are intrinsically dynamic and dynamic coupling between distant structural elements is essential for effective catalysis in modular enzymes. Therefore, distal residues are expected to have impact on enzyme function. However, few studies have investigated the role of distal residues on enzymatic catalysis. In the present study, the effects of distal residue mutations on the catalytic function of an aminoacyl-tRNA synthetase, namely, prolyl-tRNA synthase, were investigated. The present study demonstrates that distal residues significantly contribute to catalysis of the modular Escherichia coli prolyl-tRNA synthetase by maintaining intrinsic protein flexibility.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Catálisis , Dominio Catalítico , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , MutaciónRESUMEN
Quinone reductases belong to the family of flavin-dependent oxidoreductases. With the redox active cofactor, flavin adenine dinucleotide, quinone reductases are known to utilize a 'ping-pong' kinetic mechanism during catalysis in which a hydride is bounced back and forth between flavin and its two substrates. However, the continuation of this catalytic cycle requires product displacement steps, where the product of one redox half-cycle is displaced by the substrate of the next half-cycle. Using improved hybrid quantum mechanical/molecular mechanical simulations, both the catalytic hydride transfer and the product displacement reactions were studied in NRH:quinone oxidoreductase 2. Initially, the self-consistent charge-density functional tight binding theory was used to describe flavin ring and the substrate atoms, while embedded in the molecular mechanically-treated solvated active site. Then, for each step of the catalytic cycle, a further improvement of energetics was made using density functional theory-based corrections. The present study showcases an integrated interplay of solvation, protonation, and protein matrix-induced polarization as the driving force behind the thermodynamic wheel of the 'ping-pong' kinetics. Reported here is the first-principles model of the 'ping-pong' kinetics that portrays how cyclic changes in the active site polarization and dynamics govern the oscillatory hydride transfer and product displacement in this enzyme.
RESUMEN
A project-based biophysical chemistry laboratory course, which is offered to the biochemistry and molecular biology majors in their senior year, is described. In this course, the classroom study of the structure-function of biomolecules is integrated with the discovery-guided laboratory study of these molecules using computer modeling and simulations. In particular, modern computational tools are employed to elucidate the relationship between structure, dynamics, and function in proteins. Computer-based laboratory protocols that we introduced in three modules allow students to visualize the secondary, super-secondary, and tertiary structures of proteins, analyze non-covalent interactions in protein-ligand complexes, develop three-dimensional structural models (homology model) for new protein sequences and evaluate their structural qualities, and study proteins' intrinsic dynamics to understand their functions. In the fourth module, students are assigned to an authentic research problem, where they apply their laboratory skills (acquired in modules 1-3) to answer conceptual biophysical questions. Through this process, students gain in-depth understanding of protein dynamics-the missing link between structure and function. Additionally, the requirement of term papers sharpens students' writing and communication skills. Finally, these projects result in new findings that are communicated in peer-reviewed journals.
Asunto(s)
Bioquímica/educación , Proteínas/química , Proteínas/fisiología , Universidades , Laboratorios , Modelos Moleculares , Biología Molecular/educación , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The kinetics and equilibrium of the hydride transfer reaction between lumiflavin and a number of substituted quinones was studied using density functional theory. The impact of electron withdrawing/donating substituents on the redox potentials of quinones was studied. In addition, the role of these substituents on the kinetics of the hydride transfer reaction with lumiflavin was investigated in detail under the transition state (TS) theory assumption. The hydride transfer reactions were found to be more favorable for an electron-withdrawing substituent. The activation barrier exhibited a quadratic relationship with the driving force of these reactions as derived under the formalism of modified Marcus theory. The present study found a significant extent of electron delocalization in the TS that is stabilized by enhanced electrostatic, polarization, and exchange interactions. Analysis of geometry, bond-orders, and energetics revealed a predominant parallel (Leffler-Hammond) effect on the TS. Closer scrutiny reveals that electron-withdrawing substituents, although located on the acceptor ring, reduce the N-H bond order of the donor fragment in the precursor complex. Carried out in the gas-phase, this is the first ever report of a theoretical study of flavin's hydride transfer reactions with quinones, providing an unfiltered view of the electronic effect on the nuclear reorganization of donor-acceptor complexes.
RESUMEN
The RNA species SHR1 reacts with biocytin (epsilon-biotinoyl-L-lysine) in the presence of Ni(2+) or Pt(2+) to produce a metal-bridged complex that migrates more slowly than unreacted RNA in the presence of streptavidin (StrAv) on denaturing polyacrylamide gels. Mapping of reverse transcription pause sites identified G79 as a reactive nucleotide. G79 is near the 3' end of a 37 nucleotide core motif that is nearly as reactive as SHR1. SHR1 reacts with biocytin in the presence of Pt(2+) to yield a product that comigrates with the Ni(2+) product but that is much more stable, suggesting that the metal ion used in the reaction is present in the product, possibly linking the RNA to the amino acid. In support of this model, SHR1 shows a strong affinity for Ni(2+) in immobilized metal ion chromatography.
Asunto(s)
Aminoácidos/química , Níquel/química , ARN/química , Secuencia de Bases , Cartilla de ADN , Conformación de Ácido NucleicoRESUMEN
Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes.