RESUMEN
The potential of mean force simulations, widely applied in Monte Carlo or molecular dynamics simulations, are useful tools to examine the free energy variation as a function of one or more specific reaction coordinate(s) for a given system. Implementation of the potential of mean force in the simulations of biological processes, such as enzyme catalysis, can help overcome the difficulties of sampling specific regions on the energy landscape and provide useful insights to understand the catalytic mechanism. The potential of mean force simulations usually require many, possibly parallelizable, short simulations instead of a few extremely long simulations and, therefore, are fairly manageable for most research facilities. In this chapter, we provide detailed protocols for applying the potential of mean force simulations to investigate enzymatic mechanisms for several different enzyme systems.
Asunto(s)
Biocatálisis , Enzimas/metabolismo , Simulación de Dinámica Molecular , Termodinámica , Animales , Enzimas/química , Hongos/enzimología , HumanosRESUMEN
RATIONALE: Current trends for the treatment of deep partial thickness and full-thickness burns include early excision and skin grafting. In this study we retrospectively evaluated the ability of Laser Doppler Flowmetry (LDF), taken within 24h of the burn to predict: (1) burn wound depth and (2) wounds which would heal in less than 21 days. METHOD: The Laser Doppler Flowmeter (O2C, LEA Medizintechnik, Germany) was employed to non-invasively measure the cutaneous microcirculation of 173 selected areas on 28 patients who suffered burns. RESULTS: A distinct association between initial flow (<24h after burn injury) and the clinical assessment of depth of burn wounds was observed. Wounds demonstrating an initial blood flow of >100 AU were, in 93.1% of cases, correctly (positively) predicted for spontaneous healing within 21 days. A blood flow of <100 AU (negatively) predicted in 88.2%, those wounds which would not go on to heal within 21 days. Sequential measurement analysis (<24h, 3 days after injury and 6 days after injury) revealed no significant decrease in skin perfusion velocity or flow rate. CONCLUSION: LDF can provide immediate results for early determination of burn wound depth and is useful in selecting patients for conservative treatment of their burn wounds.
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Quemaduras/patología , Microcirculación , Piel/irrigación sanguínea , Cicatrización de Heridas , Adolescente , Adulto , Anciano , Femenino , Humanos , Flujometría por Láser-Doppler , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , Adulto JovenRESUMEN
This work examines the ability of semiempirical methods to describe the structure of liquid water. Particularly, the standard AM1 and PM3 methods together with recently developed PM3-PIF and PM3-MAIS parametrizations have been considered. We perform molecular dynamics simulations for a system consisting of 64 or 216 water molecules in a periodic cubic box. The whole system is described quantum mechanically. Calculations with 64 molecules have been carried out using standard SCF techniques whereas calculations with 216 molecules have been done using the divide and conquer approach. This method has also been used in one simulation case with 64 molecules for test purposes. Within this scope, the molecular dynamics program ROAR have been coupled with a linear scaling semiempirical code (DivCon) implemented in a parallel MPI version. The predicted liquid water structure using either AM1 or PM3 is shown to be very poor due to well-known limitations of these methods describing hydrogen bonds. In contrast, PM3-PIF and PM3-MAIS calculations lead to results in reasonably good agreement with experimental data. The best results for the heat of vaporization are obtained with the PM3-PIF method. The average induced dipole moment of the water molecule in the liquid is underestimated by all semiempirical techniques, which seems to be related to the NDDO approximation and to the use of minimal basis sets. A brief discussion on charge-transfer effects in liquid water is also presented.
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Agua/química , Simulación por Computador , Iones/químicaRESUMEN
Drug discovery research is increasingly dedicated to biological screening on a massive scale, which seems to imply a basic rejection of many computer-assisted techniques originally designed to add rationality to the early stages of discovery. While ever-faster and more clever 3D methodologies continue to be developed and rejected as alternatives to indiscriminant screening, simpler tools based on 2D structure have carved a stable niche in the high-throughput paradigm of drug discovery. Their staying power is due in no small part to simplicity, ease of use, and demonstrated ability to explain structure-activity data. This observation led us to wonder whether an even simpler view of structure might offer an advantage over existing 2D and 3D methods. Accordingly, we introduce 1D representations of chemical structure, which are generated by collapsing a 3D molecular model or a 2D chemical graph onto a single coordinate of atomic positions. Atoms along this coordinate are differentiated according to elemental type, hybridization, and connectivity. By aligning 1D representations to match up identical atom types, a measure of overall structural similarity is afforded. In extensive structure-activity validation tests, 1D similarities consistently outperform both Daylight 2D fingerprints and Cerius(2) pharmacophore fingerprints, suggesting that this new, simple means of representing and comparing structures may offer a significant advantage over existing tried-and-true methods.
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Diseño de Fármacos , Modelos Moleculares , Estructura Molecular , Conformación Molecular , Esteroides/química , Relación Estructura-ActividadRESUMEN
Herein, we present results from MD simulations of the Michaelis complex formed between the B. cereus zinc-beta-lactamase enzyme and benzylpenicillin. The structural and dynamical effects induced by substrate-binding, the specific role of the conserved residues, and the near attack conformers of the Michaelis complex are discussed. Quantum chemical methods (HF/6-31G* and B3LYP/6-31G*) are also applied to study the hydrolysis reaction of N-methylazetidinone catalyzed by a monozinc system consisting of the side chains of the histidine residues (His86, His88, and His149) complexed with Zn-OH and the side chains of Asp90 and His210. From this model system, we built molecular-mechanics representations of the prereactive complex and transition state configurations docked into the active site. Linear-scaling semiempirical calculations coupled with a continuum solvent model were then performed on these static models. We propose that the experimental rate data for the B. cereus enzyme is compatible with a one-step mechanism for the hydrolysis of beta-lactam substrates in which His210 acts as a proton donor.
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Bacillus cereus/enzimología , Modelos Moleculares , Penicilina G/química , Penicilinas/química , Zinc/química , beta-Lactamasas/química , Simulación por Computador , Hidrólisis , Penicilina G/metabolismo , Penicilinas/metabolismo , Conformación Proteica , Teoría Cuántica , Termodinámica , Zinc/metabolismo , beta-Lactamasas/metabolismoRESUMEN
Herein, we present results of a computational study on benzylpenicillin attachment to Lys199 of human serum albumin via an aminolysis reaction. The internal geometry of the reactive part of the system was taken from previous work at the B3LYP/6-31+G* level on the water-assisted aminolysis reaction of a penicillin model compound (Díaz, N.; Suárez, D.; Sordo, T. L. J. Am. Chem. Soc. 2000, 122, 6710--6719). The protein environment around Lys199, the 6-acylamino side chain, and the 2-methyl groups of benzylpenicillin were relaxed by carrying out geometry optimizations with a hybrid QM/MM method (PM3/AMBER). Two different mechanistic routes were explored: a one-step water-assisted process and a carboxylate and water-assisted route in which the beta-lactam carboxylate and the ancillary water molecule mediate the proton transfer from the epsilon-amino group of Lys199 to the beta-lactam leaving N atom. The corresponding energy profiles in the protein combine the B3LYP/6-31+G* and PM3 energies of the reactive subsystem (benzylpenicillin + Lys199 side chain + the ancillary water molecule) and semiempirical PM3 energies of the entire system evaluated with a "divide and conquer" linear-scaling method. It is observed that penicillin haptenation to HSA can proceed through the water-assisted concerted mechanism which is calculated to have a high energy barrier of approximately 38 kcal/mol, in agreement with the experimentally observed slow reaction kinetics.
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Lisina/química , Penicilina G/química , Penicilinas/química , Albúmina Sérica/química , Humanos , Mecánica , Modelos Moleculares , Modelos Teóricos , Penicilina G/inmunología , Penicilinas/inmunología , Teoría Cuántica , Albúmina Sérica/inmunologíaRESUMEN
Herein we report the results of a HF/6-31+G** and B3LYP/6-31+G** computational investigation of the title reaction for the peptide bond synthesis catalyzed by a single adenine. This system constitutes a reference reaction to study the basic chemical events that have been proposed to occur at the peptidyl transferase active site of ribosomes on the basis of structural determinations (Science 2000, 239, 920--931). Thus, the analysis of the geometry, charge distribution, and energetics of the critical structures involved in this title reaction yields insight into the catalytic mode of action of RNA molecules. Our computational results give further support to the hypotheses that the activated nucleotide A2451 in the ribosome acts as a base catalyst and that this role is similar to that of the His residue in the catalytic triad of serine proteases.
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Adenina/química , Proteínas Ribosómicas/síntesis química , Acetatos/química , Sitios de Unión , Catálisis , Metilaminas/química , Modelos Químicos , Péptidos/síntesis química , Teoría Cuántica , Ribosomas/químicaRESUMEN
Herein, we report molecular dynamics simulations of the mononuclear form of the Bacillus cereuszinc-beta-lactamase. We studied two different configurations which differ in the presence of a zinc-bound hydroxide or a zinc-bound water and in the protonation state of the essential His210 residue. Contacts of the catalytically important residues (Asp90, His210, Cys168, etc.) with the zinc center are characterized by the MD analyses. The nature of the Zn-OH(2) --> His210 proton transfer pathway connecting the two configurations was studied by means of QM calculations on cluster models while the relative stability of the two configurations was estimated from QM/MM calculations in the enzyme. From these results, a theoretical model for the kinetically active form of the B. cereus metalloenzyme is proposed. Some mechanistic implications and the influence of mutating the Cys168 residue are also discussed.
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Bacillus cereus/enzimología , Metaloproteínas/química , Zinc/química , beta-Lactamasas/química , Sitios de Unión , Cationes , Cristalografía por Rayos X , Enlace de Hidrógeno , Hidróxidos/química , Cinética , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Solventes , Agua/química , Compuestos de Zinc/químicaRESUMEN
The catalytic mechanism of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-directed mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent with our proposed mechanism that the 4-carboxamide of AICAR assists proton shuttling. Quantum chemical calculations on a model system of 5-amino-4-carboxamide imidazole (AICA) and formamide using the B3LYP/6-31G level of theory confirmed that the 4-carboxamide participated in the proton-shuttling mechanism. The result also indicated that the amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from the 5-amino group through the 4-carboxamide to the formamide proceeds in the same transition state. Interestingly, the calculations predicted that protonation of the N3 of the imidazole of AICA would reduce the energy barrier significantly. However, the pK(a) of the imidazole of AICAR was determined to be 3.23 +/- 0.01 by NMR titration, and AICAR is likely to bind to the enzyme with its imidazole in the free base form. An alternative pathway was suggested by modeling Lys266 to have a hydrogen-bonding interaction with the N3 of the imidazole of AICAR. Lys266 has been implicated in catalysis based on mutagenesis studies and the recent X-ray structure of AICAR Tfase. The quantum chemical calculations on a model system that contains AICA complexed with CH3NH3+ as a mimic of the Lys residue confirmed that such an interaction lowered the activation energy of the reaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its kcat is reduced by 150-fold from that of the wild type without changes in substrate and cofactor Km values. The kcat-pH profile indicated virtually no pH-dependence in the pH range 6-10.5. The results suggest that the ammonium moiety of Lys or Arg is important in catalysis, most likely acting as a general acid catalyst with a pK(a) value greater than 10.5. The H267A mutant was also prepared since His267 has been found in the active site and implicated in catalysis. The mutant enzyme showed no detectable activity while retaining its binding affinity for substrate, indicating that it plays a critical role in catalysis. We propose that His267 interacts with Lys266 to aid in the precise positioning of the general acid catalyst to the N3 of the imidazole of AICAR.
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Transferasas de Hidroximetilo y Formilo/metabolismo , Sitios de Unión , Catálisis , Fenómenos Químicos , Química Física , Concentración de Iones de Hidrógeno , Transferasas de Hidroximetilo y Formilo/química , Transferasas de Hidroximetilo y Formilo/genética , Imidazoles/química , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa , Teoría Cuántica , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Algorithmic improvements of quantum mechanical methodologies have increased our ability to study the electronic structure of fragments of a biomolecule (e.g. an enzyme active site) or entire biomolecules. Three main strategies have emerged as ways in which quantum mechanics can be applied to biomolecules. The supermolecule approach continues to be utilized, but it is slowly being replaced by the so-called coupled quantum mechanical/molecular mechanical methodologies. An exciting new direction is the continued development and application of linear-scaling quantum mechanical approaches to biomolecular systems.
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Modelos Moleculares , Proteínas/química , Teoría Cuántica , Algoritmos , Simulación por Computador , MecánicaRESUMEN
The prioritization of the screening of combinatorial libraries is an extremely important task for the rapid identification of tight binding ligands and ultimately pharmaceutical compounds. When structural information for the target is available, molecular docking is an approach that can be used for prioritization. Here, we present the initial validation of a new rapid approach to molecular docking developed for prioritizing combinatorial libraries. The algorithm is tested on 103 individual cases from the protein data bank and in nearly 90% of these cases docks the ligand to within 2.0 A of the observed binding mode. Because the mean CPU time is <5 s/mol, this approach can process hundreds of thousands of compounds per week. Furthermore, if a somewhat less thorough search is performed, the search time drops to 1 s/mol, thus allowing millions of compounds to be docked per week and tested for potential activity. Proteins 2001;43:113-124.
Asunto(s)
Técnicas Químicas Combinatorias , Proteínas/química , Sitios de Unión , Biología Computacional , Ligandos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The IIA binding site of human serum albumin (HSA) preferentially binds hydrophobic organic anions of medium size (e.g., aspirin, benzylpenicillin, warfarin, etc.) and bilirubin. This binding ability is particularly important for the distribution, metabolism, and efficacy of drugs. In addition, HSA can also covalently link to different IIA substrates owing to the presence of a highly reactive residue, Lys199, which is strategically located in the IIA site. Herein, we present results of three restrained molecular dynamics (MD) simulations of the IIA binding site on the HSA protein. From these simulations, we have determined the influence that the ionization state of the key residue, Lys199, and the nearby Lys195 has on the structure and dynamics of the IIA binding site. When Lys199 is neutral the computed average distances for the most significant interresidue contacts are in good agreement with those estimated from the X-ray coordinates. The analysis of the solvent structure and dynamics indicates that the basic form of Lys199 is likely connected to the acid form of Lys195 through a network of H-bonding water molecules with a donor --> acceptor character. The presence of these water bridges can be important for stabilizing the configuration of the IIA binding site and/or promoting a potential Lys195 --> Lys199 proton-transfer process. These results suggest that both lysine residues located in the IIA binding site of HSA, Lys195 and Lys199, could play a combined and comparable chemical role. Our simulations also give insight into the binding of bilirubin to HSA.
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Lisina/química , Albúmina Sérica/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Protones , SolventesRESUMEN
Literature data on compounds both well- and poorly-absorbed in humans were used to build a statistical pattern recognition model of passive intestinal absorption. Robust outlier detection was utilized to analyze the well-absorbed compounds, some of which were intermingled with the poorly-absorbed compounds in the model space. Outliers were identified as being actively transported. The descriptors chosen for inclusion in the model were PSA and AlogP98, based on consideration of the physical processes involved in membrane permeability and the interrelationships and redundancies between available descriptors. These descriptors are quite straightforward for a medicinal chemist to interpret, enhancing the utility of the model. Molecular weight, while often used in passive absorption models, was shown to be superfluous, as it is already a component of both PSA and AlogP98. Extensive validation of the model on hundreds of known orally delivered drugs, "drug-like" molecules, and Pharmacopeia, Inc. compounds, which had been assayed for Caco-2 cell permeability, demonstrated a good rate of successful predictions (74-92%, depending on the dataset and exact criterion used).
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Absorción Intestinal , Preparaciones Farmacéuticas/metabolismo , Transporte Biológico , Células CACO-2 , Permeabilidad de la Membrana Celular , Humanos , Modelos Biológicos , Análisis Multivariante , Reproducibilidad de los ResultadosRESUMEN
A revised generalized Born/surface area (GB/SA) continuum solvation model has been developed for water that is compatible with the Merck molecular force field (MMFF). This model gives free energies of aqueous solvation that are comparable in accuracy to the original water model when the OPLS* force field is employed. The average unsigned error in aqueous deltaGsol using the new water model and MMFF is 0.62 kcal/mol for a training set of 82 solutes compared to 1.24 kcal/mol for the original GB/SA water model and MMFF. The average unsigned errors for 47 neutral solutes outside the training set and 10 ions are 0.96 and 2.32 kcal/mol, respectively. By comparison, the average errors for the test set and ions using the original GB/SA water model are 1.76 and 5.32 kcal/mol. This revised parameter set provides a more accurate representation of aqueous solvation for use with MMFF.
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Modelos Químicos , Soluciones/química , Agua/química , Inteligencia Artificial , Metodologías Computacionales , Compuestos Orgánicos , TermodinámicaRESUMEN
In this paper we report molecular dynamics (MD) and free energy perturbation (FEP) studies carried out on enzyme-inhibitor (two hydroxamates that only differ by a carbon-carbon double bond) complexes of human fibroblast collagenase to obtain insights into the structural and energetic preferences of these inhibitors. We have developed a bonded model for the catalytic and structural zinc centers (Hoops, S. C.; et al. J. Am. Chem. Soc. 1991, 113, 8262-8270) where the electrostatic representation for this model was derived using a novel quantum-mechanical/molecular-mechanical (QM/MM) minimization procedure followed by electrostatic potential fitting. The resulting bonded model for the zinc ions was then used to generate MD trajectories for structural analysis and FEP studies. This model has satisfactorily reproduced the structural features of the active site, and furthermore, the FEP simulations gave relative free energies of binding in good agreement with experimental results. MD simulations in conjunction with the FEP are able to provide a structural explanation regarding why one hydroxamate inhibitor is favored over the other, and we are also able to make predictions about changes in the inhibitor that would enhance protein-inhibitor interactions.
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Colagenasas/química , Ácidos Hidroxámicos/química , Inhibidores de Proteasas/química , Sitios de Unión , Humanos , Metaloproteinasa 8 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Relación Estructura-Actividad , Zinc/químicaRESUMEN
In this article we characterize, from a structural point of view, all 16 members of the tubulin gene family of Caenorhabditis elegans (9 alpha-tubulins, 6 beta-tubulins, and 1 gamma-tubulin). We obtained their tertiary structures by computationally modifying the X-ray crystal structure of the pig brain alpha/beta-tubulin dimer published by Nogales et al. [Nature (London) 1998;391:199-203]. Our computational protocol involves changing the amino acids (with MIDAS; Jarvis et al., UCSF MIDAS. University of California, San Francisco, 1986) in the 3D structure of pig brain alpha/beta-tubulin dimer followed by geometry optimization with the AMBER force field (Perlman et al., AMBER 4. University of California, San Francisco, 1990). We subsequently analyze and compare the resulting structures in terms of the differences in their secondary and tertiary structures. In addition, we compare the pattern of hydrogen bonds and hydrophobic contacts in the guanosine triphosphate (GTP)-binding site for all members of the tubulin family. Our computational results show that, except for gamma-tubulin, all members of the C. elegans tubulin family have similar secondary and 3D structures and that the change in the pattern of hydrogen bonds in the GTP-binding site may be used to assess the relative stability of different alpha/beta-tubulin dimers formed by monomers of the tubulin family.
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Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Animales , Sitios de Unión , Caenorhabditis elegans/genética , Simulación por Computador , Cristalografía por Rayos X , Dimerización , Exones , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Intrones , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Programas Informáticos , Porcinos , Tubulina (Proteína)/metabolismoRESUMEN
We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic effect play in ice binding are also highlighted. For the latter it is demonstrated that the surface of ice has a clathratelike structure which favors the partitioning of hydrophobic groups to the surface of ice. It is suggested that mutations that involve the deletion of hydrophobic residues (e.g., the Leu residues) will provide insight into the role the hydrophobic effect plays in partitioning these peptides to the surface of ice.
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Lenguado/metabolismo , Glicoproteínas/metabolismo , Hielo , Secuencia de Aminoácidos , Animales , Proteínas Anticongelantes , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Lenguado/genética , Glicoproteínas/química , Glicoproteínas/genética , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Termodinámica , AguaRESUMEN
During the past decade, computer simulations of bilayers have moved from the realm of model systems to realistic systems containing tens of phospholipids along with the requisite number of water molecules hydrating the entire molecular assembly. Concomitant with the ability to model larger and larger systems, simulators have also begun to utilize more accurate numerical tools to ensure that the temperature, pressure, simulation timescales, parameter sets and long-range electrostatic interactions of bilayers are correctly accounted for in a typical molecular dynamics simulation. With these tools in hand, work has already begun to define the structure, function and dynamics of bilayer, bilayer/small molecule and bilayer/protein systems. Thus, we have reached an era in which simulators will tackle more and more detailed issues regarding complex bilayer systems.
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Simulación por Computador , Membrana Dobles de Lípidos/química , Modelos Moleculares , Anestésicos , Colesterol , PéptidosRESUMEN
The importance of maintaining the active site water network for efficient proton transfer was investigated by substituting amino acids of varying size at position 65 in carbonic anhydrase II (including four amino acids found in other CA isozymes, F, L, S, and T, and two amino acids that do not occur naturally at position 65, G and H) and measuring the rate constants for the proton transfer reactions in the variant carbonic anhydrases. Intramolecular proton transfer between zinc-bound water and H64 is significantly inhibited by the introduction of bulky residues at position 65; kcat for CO2 hydration decreases up to 26-fold, comparable to the observed decrease in intramolecular proton transfer caused by removal of H64 [Tu, C., Silverman, D. N., Forsman, C., Jonsson, B.-H., & Lindskog, S. (1989) Biochemistry 28, 7913-7918]. Intermolecular proton transfer between protonated H64 and external buffer is also inhibited, although to a lesser degree. Furthermore, an alternative proton transfer pathway, consisting of an active site solvent-mediated proton transfer from zinc-water to imidazole buffer, is inhibited in the A65F, A65L, and A65H CAII variants. Therefore, the active solvent bridge between zinc-bound water and H64 is disrupted by substitutions at position 65. The inhibition of proton transfer reactions correlates with the disruption of the crystallographically observed solvent network in the CA active site and rotation of the proton acceptor, H64 [Scolnick, L. R., & Christianson, D. W. (1996) Biochemistry 35, 16429-16434], suggesting that this solvent network, including water molecules 292, 264, and 369, or a structurally related network, forms the proton transfer pathway in CAII for both intramolecular proton transfer and stimulation of proton transfer in imidazole buffers.
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Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Sitios de Unión , Tampones (Química) , Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/genética , Humanos , Concentración de Iones de Hidrógeno , Imidazoles , Técnicas In Vitro , Cinética , Estructura Molecular , Mutagénesis Sitio-Dirigida , Protones , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solventes , Agua , Zinc/químicaRESUMEN
Molecular dynamics simulations of the tripeptide Ala-Phe-Ala-O-tert-butyl interacting with dimyristoylphosphatidylcholine lipid bilayers have been carried out. The lipid and aqueous environments of the peptide, the alkyl chain order, and the lipid and peptide dynamics have been investigated with use of density profiles, radial distribution functions, alkyl chain order parameter profiles, and time correlation functions. It appears that the alkyl chain region accommodates the peptides in the bilayer with minimal perturbation to this region. The peptide dynamics in the bilayer bound form has been compared with that of the free peptide in water. The peptide structure does not vary on the simulation time scale (of the order of hundreds of picoseconds) compared with the solution structure in which a random structure is observed.