Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 35
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Chem Theory Comput ; 2020 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-32464053

RESUMO

Electronic polarization effects have been suggested to play an important role in proton binding to titratable residues in proteins. In this work, we describe a new computational method for pKa calculations, using Monte Carlo (MC) simulations to sample protein protonation states with the Drude polarizable force field and Poisson-Boltzmann (PB) continuum electrostatic solvent model. While the most populated protonation states at the selected pH, corresponding to residues that are half-protonated at that pH, are sampled using the exact relative free energies computed with Drude particles optimized in the field of the PB implicit solvation model, we introduce an approximation for the protein polarization of low-populated protonation states to reduce the computational cost. The highly populated protonation states used to compute the polarization and pKa's are then iteratively improved until convergence. It is shown that for lysozyme, when considering 9 of the 18 titratable residues, the new method converged within two iterations with computed pKa's differing only by 0.02 pH units from pKa's estimated with the exact approach. Application of the method to predict pKa's of 94 titratable side chains in 8 proteins shows the Drude-PB model to produce physically more correct results as compared to the additive CHARMM36 (C36) force field (FF). With a dielectric constant of two assigned to the protein interior the Root Mean Square (RMS) deviation between computed and experimental pKa's is 2.07 and 3.19 pH units with the Drude and C36 models, respectively, and the RMS deviation using the Drude-PB model is relatively insensitive to the choice of the internal dielectric constant in contrast to the additive C36 model. At the higher internal dielectric constant of 20, pKa's computed with the additive C36 model converge to the results obtained with the Drude polarizable force field, indicating the need to artificially overestimate electrostatic screening in a nonphysical way with the additive FF. In addition, inclusion of both syn and anti orientations of the proton in the neutral state of acidic groups is shown to yield improved agreement with experiment. The present work, which is the first example of the use of a polarizable model for the prediction of pKa's in proteins, shows that the use of a polarizable model represents a more physically correct model for the treatment of electrostatic contributions to pKa shifts in proteins.

2.
Chempluschem ; 85(2): 334-345, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32048812

RESUMO

An efficient protocol was developed for the synthesis of π-extended isocoumarins and polycyclic aromatic hydrocarbons based on the oxidative coupling of aromatic carboxylic acids with internal alkynes catalyzed by (cyclopentadienyl)rhodium complexes. The coupling chemoselectivity strongly depends on whether Cp or the methylated Cp* ligands are used. The pentamethyl derivative [Cp*RhCl2 ]2 predominantly gives isocoumarins, while the non-methylated complex [CpRhI2 ]n produces naphthalene derivatives. The polyaromatic carboxylic acids (such as 1-naphthoic acid, 1-pyrenecarboxylic acid, fluorene-1-carboxylic acid, and dibenzofuran-4-carboxylic acid) are suitable for this approach. A mixture of Cp*H/RhCl3 can be used as a catalyst instead of [Cp*RhCl2 ]2 . The structures of 3,4-diphenylindeno[1,2-h]isochromen-1(11H)-one and 7,10-dimethyl-8,9-diphenylbenzo[pqr]tetraphene were determined by X-ray diffraction. In addition, the optical properties of the prepared compounds were studied. 7,8-Diphenyl-10H-phenaleno[1,9-gh]isochromen-10-one was employed as an emissive layer for OLED manufacturing. The OLED emits yellow-green light with a maximum intensity 1740 cd ⋅ m-2 at 15 V.

3.
Dalton Trans ; 48(46): 17298-17309, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31714560

RESUMO

The targeted design of lanthanide-based emitters for solution-processed organic light-emitting diodes (OLEDs) resulted in obtaining an NIR OLED with one of the highest efficiencies among ytterbium-based solution-processed OLEDs (30 µW W-1). The design was aimed at the combination of high luminescence efficiency with solubility and charge carrier mobility. The latter was achieved thanks to the introduction of the purposefully selected neutral ligands, which combine electron mobility and the ability to sensitize lanthanide luminescence. Besides, the HOMO and LUMO energies and charge carrier mobility of solution-processed thin films of coordination compounds were measured experimentally for the first time, and novel highly luminescent europium-based materials with PLQYs of up to 80% and purely NIR luminescent ytterbium complexes were obtained.

4.
J Comput Chem ; 40(32): 2834-2842, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31471978

RESUMO

Flavin containing molecules form a group of important cofactors that assist a wide range of enzymatic reactions. Flavins use the redox-active isoalloxazine system, which is capable of one- and two-electron transfer reactions and can exist in several protonation states. In this work, molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field were derived for biologically important flavins, including riboflavin, flavin mononucleotide, and flavin adenine dinucleotide. The model was developed for important protonation and redox states of the isoalloxazine group. The partial charges were derived using the CHARMM force field parametrization strategy, where quantum mechanics water-solute interactions are used to target optimization. In addition to monohydrate energies and geometries, electrostatic potential around the compound was used to provide additional restraints during the charge optimization. Taking into account the importance of flavin-containing molecules special attention was given to the quality of bonded terms. All bonded terms, including stiff terms and torsion angle parameters, were parametrized using exhaustive potential energy surface scans. In particular, the model reproduces well the butterfly motion of isoalloxazine in the oxidized and reduced forms as predicted by quantum mechanics in gas phase. The model quality is illustrated by simulations of four flavoproteins. Overall, the presented molecular mechanics model will be of utility to model flavin cofactors in different redox states. © 2019 Wiley Periodicals, Inc.

5.
Expert Opin Drug Discov ; 14(1): 35-46, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30477360

RESUMO

INTRODUCTION: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains the deadliest infectious disease in the world with one-third of the world's population thought to be infected. Over the years, TB mortality rate has been largely reduced; however, this progress has been threatened by the increasing appearance of multidrug-resistant Mtb. Considerable recent efforts have been undertaken to develop new generation antituberculosis drugs. Many of these attempts have relied on in silico approaches, which have emerged recently as powerful tools complementary to biochemical attempts. Areas covered: The authors review the status of pharmaceutical drug development against TB with a special emphasis on computational work. They focus on those studies that have been validated by in vitro and/or in vivo experiments, and thus, that can be considered as successful. The major goals of this review are to present target protein systems, to highlight how in silico efforts compliment experiments, and to aid future drug design endeavors. Expert opinion: Despite having access to all of the gene and protein sequences of Mtb, the search for new optimal treatments against this deadly pathogen are still ongoing. Together with the geometric growth of protein structural and sequence databases, computational methods have become a powerful technique accelerating the successful identification of new ligands.


Assuntos
Antituberculosos/administração & dosagem , Desenho de Fármacos , Tuberculose/tratamento farmacológico , Antituberculosos/farmacologia , Simulação por Computador , Desenvolvimento de Medicamentos/métodos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/microbiologia
6.
Sci Rep ; 8(1): 7031, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29728603

RESUMO

Cyclodipeptide synthases (CDPSs) form various cyclodipeptides from two aminoacyl tRNAs via a stepwise mechanism with the formation of a dipeptidyl enzyme intermediate. As a final step of the catalytic reaction, the dipeptidyl group undergoes intramolecular cyclization to generate the target cyclodipeptide product. In this work, we investigated the cyclization reaction in the cyclodipeptide synthase AlbC using QM/MM methods and free energy simulations. The results indicate that the catalytic Y202 residue is in its neutral protonated form, and thus, is not likely to serve as a general base during the reaction. We further demonstrate that the reaction relies on the conserved residue Y202 serving as a proton relay, and the direct proton transfer from the amino group to S37 of AlbC is unlikely. Calculations reveal that the hydroxyl group of tyrosine is more suitable for the proton transfer than hydroxyl groups of other amino acids, such as serine and threonine. Results also show that the residues E182, N40, Y178 and H203 maintain the correct conformation of the dipeptide needed for the cyclization reaction. The mechanism discovered in this work relies on the amino groups conserved among the entire CDPS family and, thus is expected to be universal among CDPSs.


Assuntos
Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/metabolismo , Tirosina/metabolismo , Catálise , Sequência Conservada , Ciclização , Modelos Moleculares , Mutação , Peptídeo Sintases/química , Peptídeo Sintases/genética , Conformação Proteica , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Relação Estrutura-Atividade , Tirosina/química , Tirosina/genética
7.
J Comput Chem ; 39(22): 1707-1719, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29737546

RESUMO

In this work, we have combined the polarizable force field based on the classical Drude oscillator with a continuum Poisson-Boltzmann/solvent-accessible surface area (PB/SASA) model. In practice, the positions of the Drude particles experiencing the solvent reaction field arising from the fixed charges and induced polarization of the solute must be optimized in a self-consistent manner. Here, we parameterized the model to reproduce experimental solvation free energies of a set of small molecules. The model reproduces well-experimental solvation free energies of 70 molecules, yielding a root mean square difference of 0.8 kcal/mol versus 2.5 kcal/mol for the CHARMM36 additive force field. The polarization work associated with the solute transfer from the gas-phase to the polar solvent, a term neglected in the framework of additive force fields, was found to make a large contribution to the total solvation free energy, comparable to the polar solute-solvent solvation contribution. The Drude PB/SASA also reproduces well the electronic polarization from the explicit solvent simulations of a small protein, BPTI. Model validation was based on comparisons with the experimental relative binding free energies of 371 single alanine mutations. With the Drude PB/SASA model the root mean square deviation between the predicted and experimental relative binding free energies is 3.35 kcal/mol, lower than 5.11 kcal/mol computed with the CHARMM36 additive force field. Overall, the results indicate that the main limitation of the Drude PB/SASA model is the inability of the SASA term to accurately capture non-polar solvation effects. © 2018 Wiley Periodicals, Inc.


Assuntos
Modelos Químicos , Solventes/química , Eletricidade Estática , Simulação de Dinâmica Molecular , Termodinâmica
8.
J Phys Chem Lett ; 8(15): 3452-3456, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28692296

RESUMO

Molecular dynamics simulations and DFT calculations suggest that leaching of palladium species from Pd nanoparticles in ionic liquids does not involve "naked" Pd(0) atoms or neutral ArPdX species formed by oxidative addition of arylhalides. Instead, the ionic liquid contributes largely to leaching of ionic PdX- or PdAr+ species.

9.
RNA ; 23(5): 673-682, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28143889

RESUMO

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A1-U72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A1-U72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNAfMetA1-U72, aminoacylated with methionine, in which the C1:A72 mismatch at the end of the tRNA acceptor stem has been changed to an A1-U72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A1-U72 pair adopts an unusual arrangement. A1 is in a syn conformation and forms a single H-bond interaction with U72 This interaction requires protonation of the N1 atom of A1 Moreover, the 5' phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G2 A possible role of this unusual geometry of the A1-U72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.


Assuntos
Escherichia coli/genética , RNA de Transferência de Metionina/química , Anticódon , Pareamento de Bases , Escherichia coli/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , RNA Arqueal/química , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência de Metionina/metabolismo
10.
J Phys Chem B ; 120(19): 4388-98, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27115861

RESUMO

Leucyl-tRNA synthetase catalyzes attachment of leucine amino acid to its cognate tRNA. During the second, aminoacetylation, step of the reaction, the leucyl moiety is transferred from leucyl-adenylate to the terminal A76 adenosine of tRNA. In this work, we have investigated the aminoacetylation step catalyzed by leucyl-tRNA synthase, using ab initio quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. We have modeled reaction mechanisms arising from both crystallographic studies and computational work. We invoke various groups as potential proton acceptors-namely, the phosphate and leucyl amino groups of leucyl-adenylate, the A76 base of tRNA, and the Asp80 and Glu532 residues of the protein-and consider both metal-assisted and metal-free reactions. Free energy calculations indicate that both the phosphate group of leucyl adenylate and Glu532 are not strong bases. This agrees with the results of the quantum chemical/molecular mechanical reaction path calculations which give high free energy barriers for the studied pathways involving these groups. A self-assisted mechanism with the leucyl amino group and Asp80 as proton acceptors is the most likely. Furthermore, in this mechanism the presence of a metal ion coordinated by the phosphate group and Glu532 strongly activates the reaction.


Assuntos
Leucina-tRNA Ligase/metabolismo , Acetilação , Biocatálise , Cristalografia por Raios X , Escherichia coli/enzimologia , Leucina-tRNA Ligase/química , Metais/química , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Teoria Quântica , Especificidade por Substrato , Termodinâmica , Água/química , Água/metabolismo
11.
J Comput Chem ; 37(10): 896-912, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26841080

RESUMO

More than 100 naturally occurring modified nucleotides have been found in RNA molecules, in particular in tRNAs. We have determined molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field for all these modifications using the CHARMM force field parametrization strategy. Emphasis was placed on fine tuning of the partial atomic charges and torsion angle parameters. Quantum mechanics calculations on model compounds provided the initial set of target data, and extensive molecular dynamics simulations of nucleotides and oligonucleotides in aqueous solutions were used for further refinement against experimental data. The presented parameters will allow for computational studies of a wide range of RNAs containing modified nucleotides, including the ribosome and transfer RNAs.


Assuntos
Teoria Quântica , RNA/química , Ribonucleotídeos/química , Estrutura Molecular
12.
Nucleic Acids Res ; 43(22): 10989-1002, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26438534

RESUMO

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm(5)U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity.


Assuntos
Proteínas de Saccharomyces cerevisiae/química , tRNA Metiltransferases/química , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Yarrowia/enzimologia , tRNA Metiltransferases/metabolismo
13.
Nucleic Acids Res ; 43(5): 2946-57, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25690901

RESUMO

Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eukaryotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In archaea, orthologs of eIF5 are not found and aIF2 GTPase activity is thought to be non-assisted. However, no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2γ, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2γ. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2.


Assuntos
Proteínas Arqueais/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cristalografia por Raios X , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/química , Hidrólise , Cinese , Magnésio/química , Modelos Moleculares , Mutação , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Estrutura Terciária de Proteína , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo
14.
Biochim Biophys Acta ; 1850(5): 1006-1016, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25047891

RESUMO

GTPases typically switch between an inactive, OFF conformation and an active, ON conformation when a GDP ligand is replaced by GTP. Their ON/OFF populations and activity thus depend on the stabilities of four protein complexes, two apo-protein forms, and GTP/GDP in solution. A complete characterization is usually not possible experimentally and poses major challenges for simulations. We review the most important methodological challenges and we review thermodynamic data for two GTPases involved in translation of the genetic code: archaeal Initiation Factors 2 and 5B (aIF2, aIF5B). One main challenge is the multiplicity of states and conformations, including those of GTP/GDP in solution. Another is force field accuracy, especially for interactions of GTP/GDP with co-bound divalent Mg(2+) ions. The calculation of electrostatic free energies also poses specific challenges, and requires careful protocols. For aIF2, experiments and earlier simulations showed that it is a "classic" GTPase, with distinct ON/OFF conformations that prefer to bind GTP and GDP, respectively. For aIF5B, we recently proposed a non-classic mechanism, where the ON/OFF states differ only in the protonation state of Glu81 in the nucleotide binding pocket. This model is characterized here using free energy simulations. The methodological analysis should help future studies, while the aIF2, aIF5B examples illustrate the diversity of ATPase/GTPase mechanisms. This article is part of a Special Issue entitled Recent developments of molecular dynamics.


Assuntos
Proteínas Arqueais/química , GTP Fosfo-Hidrolases/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Simulação de Dinâmica Molecular , Fatores de Iniciação de Peptídeos/química , Regulação Alostérica , Proteínas Arqueais/metabolismo , Transferência de Energia , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligantes , Magnésio/química , Fatores de Iniciação de Peptídeos/metabolismo , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , Termodinâmica
15.
Phys Chem Chem Phys ; 16(38): 20672-80, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25158763

RESUMO

Analysis of infrared spectra of palladium nanoparticles (NPs) immersed in the tri-tert-butyl-R-phosphonium-based ionic liquids (ILs) demonstrates that both cations and anions of the ILs interact with the NPs. According to quantum-chemical simulations of these interactions, the binding energy of anions to the Pd6 cluster, taken as a minimal-size model of the NPs, increases from ∼6 to ∼27 kcal mol(-1) in the order [PF6](-)≈ [BF4](-) < [Tf2N](-) < [OTf](-) < [Br](-)≪ [TFA](-). In contrast, the binding energy for all types of the [Bu(t)3PR](+) cations slightly varies at about ∼22 kcal mol(-1) only moderately depending on the choice of the R moiety (n-pentyl, 2-hydroxyethyl, 2-methoxyethyl, 2-ethoxy-2-oxoethyl). As a result, the energies of interaction between a Pd6 cluster and various ion pairs, formed by the abovementioned counter-ions, follow the order found for the anions and vary from ∼24 to ∼47 kcal mol(-1). These values are smaller than the energy of addition of a Pd atom to a Pdn cluster (∼58 kcal mol(-1)), which suggests kinetic stabilization of the NPs in phosphonium-based ILs rather than thermodynamic stabilization. The results are qualitatively similar to the trends found earlier for interactions between palladium clusters and components of imidazolium-based ILs, in spite of much larger contributions of the London dispersion forces to the binding of the [Bu(t)3PR](+) cations to the cluster (up to 80%) relative to the case of 1-R-3-methylimidazolium cations (up to 40%).


Assuntos
Líquidos Iônicos/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Modelos Químicos , Paládio/química , Fosfinas/química , Espectrofotometria Infravermelho/métodos , Simulação por Computador , Íons/química , Modelos Moleculares , Teoria Quântica , Solventes/química
16.
J Phys Chem B ; 118(17): 4505-13, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24720842

RESUMO

The enzyme citrate synthase is used by all living cells to catalyze the first step of the citric acid cycle. In this work, we have investigated the enolization and condensation steps catalyzed by citrate synthase, using ab initio (B3LYP/def2-TZVP and MP2/aug-cc-pVDZ) quantum chemical/molecular mechanical hybrid potentials in conjunction with reaction-path-location algorithms and molecular dynamics free energy simulations. The results of the latter indicate that the catalytic His238 residue is in its neutral form, and also argue strongly for the presence of a water molecule in the enzyme's catalytic center. Such a water is observed in some, but not all, of the experimentally resolved structures of the protein. The mechanism itself starts with an enolization that proceeds via an enolate intermediate rather than the enol form, which is much more unstable. This is in agreement with the results of other workers. For the condensation step, we investigated two mechanisms in which there is a direct nucleophilic attack of the enolate intermediate on the oxaloacetate carbonyl carbon, and found the one in which there is no proton transfer from the neighboring arginine to be preferred. Although this residue, Arg329, is not implicated directly in the reaction, it helps to stabilize the negative citryl-CoA formed during the condensation step.


Assuntos
Acil Coenzima A/metabolismo , Biocatálise , Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Prótons , Teoria Quântica , Termodinâmica , Água/química , Água/metabolismo
17.
Appl Environ Microbiol ; 80(13): 4003-11, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24771025

RESUMO

Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.


Assuntos
Apoenzimas/biossíntese , Arthrobacter/enzimologia , Hidrolases/biossíntese , Apoenzimas/química , Apoenzimas/genética , Simulação por Computador , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Herbicidas/metabolismo , Hidrolases/química , Hidrolases/genética , Hidrólise , Cinética , Modelos Moleculares , Proteínas Mutantes/biossíntese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Triazinas/metabolismo
18.
J Chem Theory Comput ; 10(7): 2690-709, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-26586504

RESUMO

Free energy simulations for electrostatic and charging processes in complex molecular systems encounter specific difficulties owing to the long-range, 1/r Coulomb interaction. To calculate the solvation free energy of a simple ion, it is essential to take into account the polarization of nearby solvent but also the electrostatic potential drop across the liquid-gas boundary, however distant. The latter does not exist in a simulation model based on periodic boundary conditions because there is no physical boundary to the system. An important consequence is that the reference value of the electrostatic potential is not an ion in a vacuum. Also, in an infinite system, the electrostatic potential felt by a perturbing charge is conditionally convergent and dependent on the choice of computational conventions. Furthermore, with Ewald lattice summation and tinfoil conducting boundary conditions, the charges experience a spurious shift in the potential that depends on the details of the simulation system such as the volume fraction occupied by the solvent. All these issues can be handled with established computational protocols, as reviewed here and illustrated for several small ions and three solvated proteins.

19.
RNA ; 19(9): 1218-25, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23864225

RESUMO

Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aa-tRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational G-protein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.


Assuntos
Guanosina Trifosfato/metabolismo , Histidina/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Catálise , Guanosina Trifosfato/química , Histidina/química , Histidina/genética , Hidrólise , Cinética , Simulação de Dinâmica Molecular , Mutação , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Teoria Quântica , RNA de Transferência/química , RNA de Transferência/metabolismo , Termodinâmica
20.
Phys Chem Chem Phys ; 14(36): 12544-53, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22576234

RESUMO

A common challenge in theoretical biophysics is the identification of a minimum energy path (MEP) for the rearrangement of a group of atoms from one stable configuration to another. The structure with maximum energy along the MEP approximates the transition state for the process and the energy profile itself permits estimation of the transition rates. In this work we describe a computationally efficient algorithm for the identification of minimum energy paths in complicated biosystems. The algorithm is a hybrid of the nudged elastic band (NEB) and string methods. It has been implemented in the pDynamo simulation program and tested by examining elementary steps in the reaction mechanisms of three enzymes: citrate synthase, RasGAP, and lactate dehydrogenase. Good agreement is found for the energies and geometries of the species along the reaction profiles calculated using the new algorithm and previous versions of the NEB and string techniques, and also those obtained by the common method of adiabatic exploration of the potential energy surface as a function of predefined reaction coordinates. Precisely refined structures of the saddle points along the paths may be subsequently obtained with the climbing image variant of the NEB algorithm. Directions in which the utility of the methods that we have implemented can be further improved are discussed.


Assuntos
Algoritmos , Citrato (si)-Sintase/química , L-Lactato Desidrogenase/química , Simulação de Dinâmica Molecular , Proteínas Ativadoras de ras GTPase/química , Citrato (si)-Sintase/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA