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1.
J Biomol Struct Dyn ; 39(7): 2352-2363, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32212957

RESUMEN

Design of a drug compound that can effectively bind to the M2 ion channel and block the diffusion of hydrogen ions (H+) through and inhibit influenza A virus replication is an important task. Known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus. A new class of positively charged, +2 e.u., molecules is proposed here to block diffusion of H+ ion through the M2 channel. Several drug candidates, derivatives of a lead compound (diazabicyclooctane), were proposed and investigated. Molecular dynamics of thermal fluctuations of M2 protein structure and ionization-conformation coupling of all the ionizable residues were simulated at physiological pH. The influence of the most probable mutations of key drug-binding amino acid residues in the M2 ion channel were investigated too. It is shown that the suggested new blocker has high binding affinity for the M2 channel. There are two in-channel binding sites of high affinity, the first one has H-bonds with two of four serine residues Ser-31A (B) or Ser-31C(D), and the second one has H-bonds with two of four histidine residues His-37A (B), or His-37C(D). The main advantage of the new drug molecule is the positive charge, +2 e.u., which creates a positive electrostatic potential barrier (in addition to a steric one) for a transfer of H+ ion through M2 channel and may serve as an effective anti-influenza A virus drug.Communicated by Ramaswamy H. Sarma.


Asunto(s)
Gripe Humana , Preparaciones Farmacéuticas , Antivirales/farmacología , Antivirales/uso terapéutico , Humanos , Gripe Humana/tratamiento farmacológico , Canales Iónicos/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo
2.
J Phys Chem B ; 123(45): 9546-9556, 2019 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-31633353

RESUMEN

In the repair of the damage to bases, human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a key participant via the DNA base excision repair pathway. APE1 cleaves AP sites in DNA, which are potentially cytotoxic and highly mutagenic if left unrepaired. According to existing structural data, this enzyme's active site contains many polar amino acid residues, which form extensive contacts with a DNA substrate. A few alternative catalytic mechanisms of the phosphodiester bond hydrolysis by APE1 have been reported. Here, the kinetics of conformational changes of the enzyme and of DNA substrate molecules were studied during the recognition and cleavage of the abasic site in the pH range from 5.5 to 9.0 using stopped-flow fluorescence techniques. The activity of APE1 increased with an increase in pH because of acceleration of the rates of catalytic complex formation and of the catalytic reaction. Molecular dynamics simulation uncovered a significant increase in the pKa of His-309 located in the active site of the enzyme. This finding revealed that the observed enhancement of enzymatic activity with pH could be associated with deprotonation of not only Tyr-171 but also His-309. The obtained data allowed us to hypothesize that the ionized state of these residues could be a molecular switch between the alternative catalytic mechanisms, which involve different functionalities of these residues throughout the reaction.


Asunto(s)
Aminoácidos/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN/metabolismo , Biocatálisis , Dominio Catalítico , ADN/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Humanos , Concentración de Iones de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica
3.
Molecules ; 24(17)2019 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-31466351

RESUMEN

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme-DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme-DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.


Asunto(s)
ADN/metabolismo , Uracil-ADN Glicosidasa/química , Uracil-ADN Glicosidasa/metabolismo , Sustitución de Aminoácidos , Arginina/genética , Dominio Catalítico , Daño del ADN , Histidina/genética , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Fenilalanina/genética , Unión Proteica , Uracil-ADN Glicosidasa/genética
4.
J Phys Chem B ; 123(23): 4878-4887, 2019 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-31117610

RESUMEN

Human 8-oxoguanine-DNA glycosylase (hOGG1) possesses very high specificity for 8-oxoguanine (oxoG), even though this damaged base differs from normal guanine by only two atoms. Our aim was to determine the roles of certain catalytically important amino acid residues in the hOGG1 enzymatic pathway and describe their involvement in the mechanism of DNA lesion recognition. Molecular dynamic simulation and pre-steady-state fluorescence kinetics were performed to analyze the conformational behavior of wild-type hOGG1 and mutants G42S, D268A, and K249Q, as well as damaged and undamaged DNA. A loss of electrostatic interactions in the K249Q mutant leads to the disruption of specific contacts in the active site of the enzyme and the loss of catalytic activity. The absence of residue Asp-268 abrogates the ability of the enzyme to fully flip out the oxoG base from the double helix, thereby disrupting proper positioning of the damaged base in the active site. Furthermore, substitution of Gly-42 with Ser, which forms a damage-specific H-bond with the N7 atom of the oxoG base, creates a stable H-bond between N7 of undamaged G and Oγ of Ser-42. Nevertheless, positioning of the undamaged base in the active site is unsuitable for catalytic hydrolysis of the N-glycosidic bond.


Asunto(s)
Aminoácidos/metabolismo , ADN Glicosilasas/metabolismo , ADN/metabolismo , Aminoácidos/química , Sitios de Unión , ADN/química , Daño del ADN , ADN Glicosilasas/genética , Fluorescencia , Humanos , Cinética , Simulación de Dinámica Molecular , Mutación , Conformación Proteica
5.
PeerJ Phys Chem ; 12019 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-34079958

RESUMEN

There is abundant theoretical evidence indicating that a mirror image of Protein A may occur during the protein folding process. However, as to whether such mirror image exists in solution is an unsolved issue. Here we provide outline of an experimental design aimed to detect the mirror image of Protein A in solution. The proposal is based on computational simulations indicating that the use of a mutant of protein A, namely Q10H, could be used to detect the mirror image conformation in solution. Our results indicate that the native conformation of the protein A should have a pKa, for the Q10H mutant, at ≈6.2, while the mirror-image conformation should have a pKa close to ≈7.3. Naturally, if all the population is in the native state for the Q10H mutant, the pKa should be ≈6.2, while, if all are in the mirror-image state, it would be ≈7.3, and, if it is a mixture, the pKa should be largerthan 6.2, presumably in proportion to the mirror population. In addition, evidence is provided indicating the tautomeric distribution of H10 must also change between the native and mirror conformations. Although this may not be completely relevant for the purpose of determining whether the protein A mirror image exists in solution, it could provide valuable information to validate the pKa findings. We hope this proposal will foster experimental work on this problem either by direct application of our proposed experimental design or serving as inspiration and motivation for other experiments.

6.
J Biomol Struct Dyn ; 36(12): 3094-3105, 2018 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28884632

RESUMEN

A recently introduced electrostatic-based method to determine the pKa values of ionizable residues and fractions of ionized and tautomeric forms of histidine (His) and acid residues in proteins, at a given fixed pH, is applied here to the analysis of a His-rich protein, namely Loligo vulgaris (pdb id 1E1A), a 314-residue all-ß protein. The average tautomeric fractions for the imidazole ring of each of the six histidines in the sequence were computed using an approach that includes, but is not limited to, molecular dynamic simulations coupled with calculations of the ionization states for all 94 ionizable residues of protein 1E1A in water at pH 6.5 and 300 K. The electrostatic-calculated tautomeric fractions of the imidazole ring of His were compared with predictions obtained from an existent NMR-based methodology. Our results indicate that: (i) the averaged electrostatic-based tautomeric predictions for the imidazole ring of all histidines of Loligo vulgaris are dominated by the Nε2-H rather than the Nδ1-H form, although such preferences from the NMR-based methodology are not so well defined; (ii) the computed average absolute difference between the electrostatic- and the NMR-based tautomeric predictions among all six histidines vary among 0% to 17%; (iii) for the His showing the largest fraction of the neutral form (81%), the absolute difference between the NMR- and electrostatic-based computed tautomeric predictions is only 3%; and (iv) the tautomeric predictions for the imidazole ring of His computed with the NMR-based methodology are stable within a certain, well-defined, range of variations of a tautomer-related parameter.


Asunto(s)
Histidina/química , Imidazoles/química , Loligo/química , Animales , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Simulación del Acoplamiento Molecular , Proteínas/química , Electricidad Estática
7.
J Biomol Struct Dyn ; 36(3): 561-574, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-28132613

RESUMEN

A computational method, to predict the pKa values of the ionizable residues Asp, Glu, His, Tyr, and Lys of proteins, is presented here. Calculation of the electrostatic free-energy of the proteins is based on an efficient version of a continuum dielectric electrostatic model. The conformational flexibility of the protein is taken into account by carrying out molecular dynamics simulations of 10 ns in implicit water. The accuracy of the proposed method of calculation of pKa values is estimated from a test set of experimental pKa data for 297 ionizable residues from 34 proteins. The pKa-prediction test shows that, on average, 57, 86, and 95% of all predictions have an error lower than 0.5, 1.0, and 1.5 pKa units, respectively. This work contributes to our general understanding of the importance of protein flexibility for an accurate computation of pKa, providing critical insight about the significance of the multiple neutral states of acid and histidine residues for pKa-prediction, and may spur significant progress in our effort to develop a fast and accurate electrostatic-based method for pKa-predictions of proteins as a function of pH.


Asunto(s)
Simulación de Dinámica Molecular , Muramidasa/química , Proteínas/química , Termodinámica , Entropía , Concentración de Iones de Hidrógeno , Cinética , Conformación Proteica , Electricidad Estática
8.
J Phys Chem B ; 119(49): 15221-34, 2015 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-26569147

RESUMEN

A molecular dynamics simulation approach was applied for the prediction of the thermal stability of oligonucleotide duplexes. It was shown that the enthalpy of the DNA/DNA complex formation could be calculated using this approach. We have studied the influence of various simulation parameters on the secondary structure and the hybridization enthalpy value of Dickerson-Drew dodecamer. The optimal simulation parameters for the most reliable prediction of the enthalpy values were determined. The thermodynamic parameters (enthalpy and entropy changes) of a duplex formation were obtained experimentally for 305 oligonucleotides of various lengths and GC-content. The resulting database was studied with molecular dynamics (MD) simulation using the optimized simulation parameters. Gibbs free energy changes and the melting temperatures were evaluated using the experimental correlation between enthalpy and entropy changes of the duplex formation and the enthalpy values calculated by the MD simulation. The average errors in the predictions of enthalpy, the Gibbs free energy change, and the melting temperature of oligonucleotide complexes were 11%, 10%, and 4.4 °C, respectively. We have shown that the molecular dynamics simulation gives a possibility to calculate the thermal stability of native DNA/DNA complexes a priori with an unexpectedly high accuracy.


Asunto(s)
Temperatura , Termodinámica , Simulación de Dinámica Molecular , Oligonucleótidos/química
9.
J Comput Chem ; 34(4): 319-25, 2013 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-23047307

RESUMEN

Most of existing software for analysis of molecular dynamics (MD) simulation results is based on command-line, script-guided processes that require the researchers to have an idea about programming language constructions used, often applied to the one and only product. Here, we describe an open-source cross-platform program, MD Trajectory Reader and Analyzer (MDTRA), that performs a large number of MD analysis tasks assisted with a graphical user interface. The program has been developed to facilitate the process of search and visualization of results. MDTRA can handle trajectories as sets of protein data bank files and presents tools and guidelines to convert some other trajectory formats into such sets. The parameters analyzed by MDTRA include interatomic distances, angles, dihedral angles, angles between planes, one-dimensional and two-dimensional root-mean-square deviation, solvent-accessible area, and so on. As an example of using the program, we describe the application of MDTRA to analyze the MD of formamidopyrimidine-DNA glycosylase, a DNA repair enzyme from Escherichia coli.


Asunto(s)
ADN-Formamidopirimidina Glicosilasa/química , Escherichia coli/enzimología , Simulación de Dinámica Molecular , Programas Informáticos , Gráficos por Computador , Interfaz Usuario-Computador
10.
J Comput Chem ; 33(8): 832-42, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22278814

RESUMEN

An advanced implicit solvent model of water-proton bath for protein simulations at constant pH is presented. The implicit water-proton bath model approximates the potential of mean force of a protein in water solvent in a presence of hydrogen ions. Accurate and fast computational implementation of the implicit water-proton bath model is developed using the continuum electrostatic Poisson equation model for calculation of ionization equilibrium and the corrected MSR6 generalized Born model for calculation of the electrostatic atom-atom interactions and forces. Molecular dynamics (MD) method for protein simulation in the potential of mean force of water-proton bath is developed and tested on three proteins. The model allows to run MD simulations of proteins at constant pH, to calculate pH-dependent properties and free energies of protein conformations. The obtained results indicate that the developed implicit model of water-proton bath provides an efficient way to study thermodynamics of biomolecular systems as a function of pH, pH-dependent ionization-conformation coupling, and proton transfer events.


Asunto(s)
Simulación de Dinámica Molecular , Muramidasa/química , Ribonucleasas/química , Agua/química , Animales , Bovinos , Pollos , Concentración de Iones de Hidrógeno , Protones , Termodinámica
11.
Biochemistry ; 51(6): 1306-21, 2012 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-22243137

RESUMEN

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from exposure to UV light, ionizing radiation, alkylating agents, and oxygen radicals. In human cells, AP endonuclease 1 (APE1) recognizes this mutagenic lesion and initiates its repair via a specific incision of the phosphodiester backbone 5' to the AP site. We have investigated a detailed mechanism of APE1 functioning using fluorescently labeled DNA substrates. A fluorescent adenine analogue, 2-aminopurine, was introduced into DNA substrates adjacent to the abasic site to serve as an on-site reporter of conformational transitions in DNA during the catalytic cycle. Application of a pre-steady-state stopped-flow technique allows us to observe changes in the fluorescence intensity corresponding to different stages of the process in real time. We also detected an intrinsic Trp fluorescence of the enzyme during interactions with 2-aPu-containing substrates. Our data have revealed a conformational flexibility of the abasic DNA being processed by APE1. Quantitative analysis of fluorescent traces has yielded a minimal kinetic scheme and appropriate rate constants consisting of four steps. The results obtained from stopped-flow data have shown a substantial influence of the 2-aPu base location on completion of certain reaction steps. Using detailed molecular dynamics simulations of the DNA substrates, we have attributed structural distortions of AP-DNA to realization of specific binding, effective locking, and incision of the damaged DNA. The findings allowed us to accurately discern the step that corresponds to insertion of specific APE1 amino acid residues into the abasic DNA void in the course of stabilization of the precatalytic complex.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN/química , Simulación de Dinámica Molecular , 2-Aminopurina/química , Catálisis , ADN/metabolismo , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Colorantes Fluorescentes , Humanos , Ácidos Nucleicos Heterodúplex/química , Oligodesoxirribonucleótidos/química , Conformación Proteica , Mapeo de Interacción de Proteínas , Espectrometría de Fluorescencia/métodos , Especificidad por Sustrato , Triptófano/química
12.
Adv Protein Chem Struct Biol ; 85: 281-322, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21920327

RESUMEN

Modern implicit solvent models for macromolecular simulations in water-proton bath are considered. The fundamental quantity that implicit models approximate is the solute potential of mean force, which is obtained by averaging over solvent degrees of freedom. The implicit solvent models suggest practical ways to calculate free energies of macromolecular conformations taking into account equilibrium interactions with water solvent and proton bath, while the explicit solvent approach is unable to do that due to the need to account for a large number of solvent degrees of freedom. The most advanced realizations of the implicit continuum models by different research groups are discussed, their accuracy are examined, and some applications of the implicit solvent models to macromolecular modeling, such as free energy calculations, protein folding, and constant pH molecular dynamics are highlighted.


Asunto(s)
Biología Computacional , Simulación de Dinámica Molecular , Proteínas/química , Solventes/química , Termodinámica , Agua/química , Concentración de Iones de Hidrógeno , Conformación Proteica
13.
Proc Natl Acad Sci U S A ; 108(14): 5602-7, 2011 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-21422292

RESUMEN

A method is proposed to determine the fraction of the tautomeric forms of the imidazole ring of histidine in proteins as a function of pH, provided that the observed and chemical shifts and the protein structure, or the fraction of H(+) form, are known. This method is based on the use of quantum chemical methods to compute the (13)C NMR shieldings of all the imidazole ring carbons ((13)C(γ), , and ) for each of the two tautomers, N(δ1)-H and N(ε2)-H, and the protonated form, H(+), of histidine. This methodology enabled us (i) to determine the fraction of all the tautomeric forms of histidine for eight proteins for which the and chemical shifts had been determined in solution in the pH range of 3.2 to 7.5 and (ii) to estimate the fraction of tautomeric forms of eight histidine-containing dipeptide crystals for which the chemical shifts had been determined by solid-state (13)C NMR. Our results for proteins indicate that the protonated form is the most populated one, whereas the distribution of the tautomeric forms for the imidazole ring varies significantly among different histidines in the same protein, reflecting the importance of the environment of the histidines in determining the tautomeric forms. In addition, for ∼70% of the neutral histidine-containing dipeptides, the method leads to fairly good agreement between the calculated and the experimental tautomeric form. Coexistence of different tautomeric forms in the same crystal structure may explain the remaining 30% of disagreement.


Asunto(s)
Histidina/química , Imidazoles/química , Proteínas/química , Isótopos de Carbono , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Químicos
14.
J Comput Chem ; 31(5): 1080-92, 2010 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-19821514

RESUMEN

This study describes the development of a new blind hierarchical docking method, bhDock, its implementation, and accuracy assessment. The bhDock method uses two-step algorithm. First, a comprehensive set of low-resolution binding sites is determined by analyzing entire protein surface and ranked by a simple score function. Second, ligand position is determined via a molecular dynamics-based method of global optimization starting from a small set of high ranked low-resolution binding sites. The refinement of the ligand binding pose starts from uniformly distributed multiple initial ligand orientations and uses simulated annealing molecular dynamics coupled with guided force-field deformation of protein-ligand interactions to find the global minimum. Assessment of the bhDock method on the set of 37 protein-ligand complexes has shown the success rate of predictions of 78%, which is better than the rate reported for the most cited docking methods, such as AutoDock, DOCK, GOLD, and FlexX, on the same set of complexes.


Asunto(s)
Proteínas/química , Proteínas/metabolismo , Sitios de Unión , Ligandos , Simulación de Dinámica Molecular , Unión Proteica
15.
Proteins ; 77(1): 38-51, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19384995

RESUMEN

Availability of energy functions which can discriminate native-like from non-native protein conformations is crucial for theoretical protein structure prediction and refinement of low-resolution protein models. This article reports the results of benchmark tests for scoring functions based on two all-atom ECEPP force fields, that is, ECEPP/3 and ECEPP05, and two implicit solvent models for a large set of protein decoys. The following three scoring functions are considered: (i) ECEPP05 plus a solvent-accessible surface area model with the parameters optimized with a set of protein decoys (ECEPP05/SA); (ii) ECEPP/3 plus the solvent-accessible surface area model of Ooi et al. (Proc Natl Acad Sci USA 1987;84:3086-3090) (ECEPP3/OONS); and (iii) ECEPP05 plus an implicit solvent model based on a solution of the Poisson equation with an optimized Fast Adaptive Multigrid Boundary Element (FAMBEpH) method (ECEPP05/FAMBEpH). Short Monte Carlo-with-Minimization (MCM) simulations, following local energy minimization, are used as a scoring method with ECEPP05/SA and ECEPP3/OONS potentials, whereas energy calculation is used with ECEPP05/FAMBEpH. The performance of each scoring function is evaluated by examining its ability to distinguish between native-like and non-native protein structures. The results of the tests show that the new ECEPP05/SA scoring function represents a significant improvement over the earlier ECEPP3/OONS version of the force field. Thus, it is able to rank native-like structures with C(alpha) root-mean-square-deviations below 3.5 A as lowest-energy conformations for 76% and within the top 10 for 87% of the proteins tested, compared with 69 and 80%, respectively, for ECEPP3/OONS. The use of the FAMBEpH solvation model, which provides a more accurate description of the protein-solvent interactions, improves the discriminative ability of the scoring function to 89%. All failed tests in which the native-like structures cannot be discriminated as those with low energy, are due to omission of protein-protein interactions. The results of this study represent a benchmark in force-field development, and may be useful for evaluation of the performance of different force fields.


Asunto(s)
Biología Computacional/métodos , Modelos Químicos , Proteínas/química , Solventes/química , Algoritmos , Conformación Proteica , Electricidad Estática , Termodinámica
16.
J Phys Chem B ; 112(35): 11122-36, 2008 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-18683966

RESUMEN

A fast and accurate method to compute the total solvation free energies of proteins as a function of pH is presented. The method makes use of a combination of approaches, some of which have already appeared in the literature; (i) the Poisson equation is solved with an optimized fast adaptive multigrid boundary element (FAMBE) method; (ii) the electrostatic free energies of the ionizable sites are calculated for their neutral and charged states by using a detailed model of atomic charges; (iii) a set of optimal atomic radii is used to define a precise dielectric surface interface; (iv) a multilevel adaptive tessellation of this dielectric surface interface is achieved by using multisized boundary elements; and (v) 1:1 salt effects are included. The equilibrium proton binding/release is calculated with the Tanford-Schellman integral if the proteins contain more than approximately 20-25 ionizable groups; for a smaller number of ionizable groups, the ionization partition function is calculated directly. The FAMBE method is tested as a function of pH (FAMBE-pH) with three proteins, namely, bovine pancreatic trypsin inhibitor (BPTI), hen egg white lysozyme (HEWL), and bovine pancreatic ribonuclease A (RNaseA). The results are (a) the FAMBE-pH method reproduces the observed pK a's of the ionizable groups of these proteins within an average absolute value of 0.4 p K units and a maximum error of 1.2 p K units and (b) comparison of the calculated total pH-dependent solvation free energy for BPTI, between the exact calculation of the ionization partition function and the Tanford-Schellman integral method, shows agreement within 1.2 kcal/mol. These results indicate that calculation of total solvation free energies with the FAMBE-pH method can provide an accurate prediction of protein conformational stability at a given fixed pH and, if coupled with molecular mechanics or molecular dynamics methods, can also be used for more realistic studies of protein folding, unfolding, and dynamics, as a function of pH.


Asunto(s)
Proteínas/química , Solventes/química , Algoritmos , Animales , Bovinos , Conductividad Eléctrica , Concentración de Iones de Hidrógeno , Modelos Moleculares , Muramidasa/química , Conformación Proteica , Ribonucleasa Pancreática/química , Sensibilidad y Especificidad , Electricidad Estática , Termodinámica , Factores de Tiempo
17.
Biochimie ; 90(11-12): 1624-36, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18585432

RESUMEN

This study is directed towards an important problem concerning the organization of the peptidyl transferase center (PTC) on the mammalian ribosome that cannot be studied by X-ray analysis since crystals of 80S ribosomes are still unavailable. Here, we investigated the arrangement of the 3'-end of tRNA in the 80S ribosomal A and P sites using a tRNA(Asp) analogue that bears a 4-thiouridine (s(4)U) attached to the 3'-terminal adenosine. It was shown that an additional nucleotide s(4)U77 on the 3'-end does not impede codon-dependent binding of the tRNA to the A and P sites of 80S ribosome. Mild UV-irradiation of the ribosomal complexes containing a short appropriately designed mRNA and the tRNA analogue resulted in cross-linking of the analogue exclusively to 28S rRNA. The cross-linking site was detected in the 4302-4540 fragment of the 28S rRNA which belongs to the highly conserved domain V that in prokaryotic ribosomes is involved in the formation of the PTC. Nucleotides cross-linked to the tRNA analogue were determined by means of reverse transcription. A comparison of the results obtained with a dynamic model of mutual arrangement of s(4)U77 of the A site tRNA and nucleotides of 23S rRNA built on the basis of an atomic model for the prokaryotic PTC led to the conclusion that environments of the tRNA 3'-terminus in prokaryotic and eukaryotic ribosomes share a significant extent of similarity, although pronounced differences are also detectable.


Asunto(s)
Conformación de Ácido Nucleico , ARN de Transferencia de Aspártico/química , ARN de Transferencia de Fenilalanina/química , Ribosomas/metabolismo , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , ARN Mensajero/química , ARN Ribosómico 23S/química , ARN Ribosómico 28S/química
18.
Proteins ; 61(1): 56-68, 2005 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-16080152

RESUMEN

Interest centers here on whether the use of a fixed charge distribution of a protein solute, or a treatment that considers proton-binding equilibria by solving the Poisson equation, is a better approach to discriminate native from non-native conformations of proteins. In this analysis of the charge distribution of 7 proteins, we estimate the solvation free energy contribution to the total free energy by exploring the 2(zeta) possible ionization states of the whole molecule, with zeta being the number of ionizable groups in the amino acid sequence, for every conformation in the ensembles of 7 proteins. As an additional consideration of the role of electrostatic interactions in determining the charge distribution of native folds, we carried out a comparison of alternative charge assignment models for the ionizable residues in a set of 21 native-like proteins. The results of this work indicate that (1) for 6 out of 7 proteins, estimation of solvent polarization based on the Generalized Born model with a fixed charge distribution provides the optimal trade-off between accuracy, with respect to the Poisson equation, and speed when compared to the accessible surface area model; for the seventh protein, consideration of all possible ionization states of the whole molecule appears to be crucial to discriminate the native from non-native conformations; (2) significant differences in the degree of ionization and hence the charge distribution for native folds are found between the different charge models examined; (3) the stability of the native state is determined by a delicate balance of all the energy components, and (4) conformational entropy, and hence the dynamics of folding, may play a crucial role for a successful ab initio protein folding prediction.


Asunto(s)
Proteínas/química , Proteínas/metabolismo , Protones , Iones/química , Modelos Biológicos , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína
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