RESUMO
Bacterial photosynthetic membranes, also known as chromatophores, are tightly packed with integral membrane proteins that work together to carry out photosynthesis. Chromatophores display a wide range of cellular morphologies; spherical, tubular, and lamellar chromatophores have all been observed in different bacterial species, or with different protein constituents. Through recent computational modeling and simulation, it has been demonstrated that the light-harvesting complexes abundant in chromatophores induce local membrane curvature via multiple mechanisms. These protein complexes assemble to generate a global curvature and sculpt the chromatophores into various cellular-scale architectures.
Assuntos
Cromatóforos/química , Proteínas de Bactérias/química , Complexos de Proteínas Captadores de Luz/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Fotossíntese , Estrutura Terciária de ProteínaRESUMO
Hybrid computational methods for combining structural data from different sources and resolutions are becoming an essential part of structural biology, especially as the field moves toward the study of large macromolecular assemblies. We have developed the molecular dynamics flexible fitting (MDFF) method for combining high-resolution atomic structures with cryo-electron microscopy (cryo-EM) maps, that results in atomic models representing the conformational state captured by cryo-EM. The method has been applied successfully to the ribosome, a ribonucleoprotein complex responsible for protein synthesis. MDFF involves a molecular dynamics simulation in which a guiding potential, based on the cryo-EM map, is added to the standard force field. Forces proportional to the gradient of the density map guide an atomic structure, available from X-ray crystallography, into high-density regions of a cryo-EM map. In this paper we describe the necessary steps to set up, run, and analyze MDFF simulations and the software packages that implement the corresponding functionalities.
Assuntos
Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , RNA Catalítico/química , Biofísica/métodos , Simulação por Computador , Modelos Moleculares , Modelos Estatísticos , Conformação Molecular , Ácidos Nucleicos/química , Estrutura Secundária de Proteína , Ribossomos/metabolismo , Software , Solventes/química , Fatores de TempoRESUMO
The C57BL/6J strain of laboratory mice is a popular subject for studies of diet-induced obesity and diabetes given its propensity for developing obesity and glucose intolerance when placed on high-fat diet. High-fat diet leads to much lower weight gain in young adult BALB/cJ mice, which appear to be protected from many of the metabolic effects of high-fat diet observed in C57BL/6J mice. In this report, the effects of diet and timing of feeding on body weight, food intake, glucose tolerance, and stress-induced corticosterone and blood glucose responses were assessed in male C57BL/6J and BALB/cJ mice. Lower glucose tolerance was observed in low-fat diet-fed C57BL/6J than BALB/cJ mice at four times sampled across the circadian cycle. Ad libitum high-fat diet increased the amount of daytime eating behavior and led to impaired glucose regulation in C57BL/6J but not BALB/cJ mice. Restricting food availability to either daytime or nighttime did not prevent overall body weight gain, but restricting feeding to nighttime (but not daytime) did prevent the significant increase in perigonadal fat pad mass produced by high-fat diet in C57BL/6J mice. Baseline corticosterone levels at their typical daily peak near onset of daily activity were blunted in both strains of mice after 8 weeks on high-fat diet, without corresponding differences in baseline glucose levels. Restraint stress-induced increases in corticosterone were exaggerated in C57BL/6J mice on high-fat diet, with concomitant increases in blood glucose. Paradoxically, stress-induced corticosterone responses were even more exaggerated in BALB/cJ mice yet with significantly blunted glucose responses compared to C57BL/6J mice, regardless of diet, indicating that corticosterone does not have equivalent glucogenic effects in young adult male BALB/cJ and C57BL/6J mice on high-fat diet. These results document considerable strain differences that may provide means for elucidating the mechanisms involved in diet-induced obesity, while highlighting the need to consider these strain differences when extending the results of mouse studies toward the human condition.
Assuntos
Dieta Hiperlipídica , Ingestão de Alimentos , Glucocorticoides/metabolismo , Intolerância à Glucose/metabolismo , Tecido Adiposo/anatomia & histologia , Animais , Glicemia/análise , Corticosterona/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Atividade Motora , Especificidade da Espécie , Estresse Psicológico/metabolismo , Aumento de PesoRESUMO
In purple bacteria, photosynthesis is carried out on large indentations of the bacterial plasma membrane termed chromatophores. Acting as primitive organelles, chromatophores are densely packed with the membrane proteins necessary for photosynthesis, including light harvesting complexes LH1 and LH2, reaction center (RC), and cytochrome bc(1). The shape of chromatophores is primarily dependent on species, and is typically spherical or flat. How these shapes arise from the protein-protein and protein-membrane interactions is still unknown. Now, using molecular dynamics simulations, we have observed the dynamic curvature of membranes caused by proteins in the chromatophore. A membrane-embedded array of LH2s was found to relax to a curved state, both for LH2 from Rps. acidophila and a homology-modeled LH2 from Rb. sphaeroides. A modeled LH1-RC-PufX dimer was found to develop a bend at the dimerizing interface resulting in a curved shape as well. In contrast, the bc(1) complex, which has not been imaged yet in native chromatophores, did not induce a preferred membrane curvature in simulation. Based on these results, a model for how the different photosynthetic proteins influence chromatophore shape is presented.
Assuntos
Cromatóforos Bacterianos/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Fotossíntese , Rodopseudomonas/metabolismo , Sequência de Aminoácidos , Cromatóforos Bacterianos/metabolismo , Proteínas de Bactérias/química , Dimerização , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
The development of class- and isoform-selective histone deacetylase (HDAC) inhibitors is highly desirable for the study of the complex interactions of these proteins central to transcription regulation as well as for the development of selective HDAC inhibitors as drugs in epigenetics. To provide a structural basis for the rational design of such inhibitors, a combined computational and experimental study of inhibition of three different histone deacetylase isoforms, HDAC1, -6, and -8, with three different hydroxamate inhibitors is reported. While SAHA was found to be unselective for the inhibition of class I and class II HDACs, the other inhibitors were found to be selective toward class II HDACs. Molecular dynamics simulations indicate that this selectivity is caused by both the overall shape of the protein surface leading to the active site and specific interactions of an aspartate residue in a polar loop and two phenylalanines and a methionine in a nonpolar loop. Monitoring the specific interactions as a function of the simulation time identifies a key sulfur-pi interaction. The implications of the structural motifs for the design of class II-selective HDAC inhibitors are discussed.
Assuntos
Inibidores de Histona Desacetilases , Modelos Moleculares , Proteínas Repressoras/antagonistas & inibidores , Anilidas/química , Cristalografia por Raios X , Histona Desacetilase 1 , Desacetilase 6 de Histona , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Conformação Proteica , Proteínas Repressoras/química , Relação Estrutura-Atividade , VorinostatRESUMO
ATP hydrolysis is a key reaction in living cells that drives many cellular processes. The reaction, which involves gamma phosphate cleavage from ATP, converting it to ADP, has been suggested to occur via an associative or dissociative mechanism dependent upon the surrounding environment. Prior quantum chemical studies suffered from short simulation timescales failing to capture free energy contributions due to relaxation of the surrounding aqueous environment. We have developed a highly parallelized QM/MM implementation in the NAMD and OpenAtom simulation packages, using the dual grid, dual length scale method for combined plane-wave and Eular exponential spline-based QM/MM simulations. This approach, using message-driven parallel quantum and classical dynamics, permits sufficient timescale simulations for quantum chemical events such as ATP hydrolysis, and is found to accurately and reliably include the free energy contributions of solvent relaxation to hydrolysis. In this paper we describe the application of the dual grid, dual length plane-wave-based QM/MM method to study both the associative and dissociative mechanisms of ATP hydrolysis, accounting for the free energy contribution from solvent relaxation, as well as for the key role of Mg(2+) in the reaction.
RESUMO
Accelerated molecular dynamics (aMD) is an enhanced-sampling method that improves the conformational space sampling by reducing energy barriers separating different states of a system. Here we present the implementation of aMD in the parallel simulation program NAMD. We show that aMD simulations performed with NAMD have only a small overhead compared with classical MD simulations. Through example applications to the alanine dipeptide, we discuss the choice of acceleration parameters, the interpretation of aMD results, as well as the advantages and limitations of the aMD method.
RESUMO
Experimental studies of protein folding processes are frequently hampered by the fact that only low resolution structural data can be obtained with sufficient temporal resolution. Molecular dynamics simulations offer a complementary approach, providing extremely high resolution spatial and temporal data on folding processes. The effectiveness of such simulations is currently hampered by continuing questions regarding the ability of molecular dynamics force fields to reproduce the true potential energy surfaces of proteins, and ongoing difficulties with obtaining sufficient sampling to meaningfully comment on folding mechanisms. We review recent progress in the simulation of three common model systems for protein folding, and discuss how recent advances in technology and theory are allowing protein folding simulations to address their current shortcomings.
RESUMO
Regulatory nascent chains interact with the ribosomal exit tunnel and modulate their own translation. To characterize nascent chain recognition by the ribosome at the atomic level, extensive molecular dynamics simulations of TnaC, the leader peptide of the tryptophanase operon, inside the exit tunnel were performed for an aggregate time of 2.1 mus. The simulations, complemented by quantum chemistry calculations, suggest that the critical TnaC residue W12 is recognized by the ribosome via a cation-pi interaction, whereas TnaC's D16 forms salt bridges with ribosomal proteins. The simulations also show that TnaC-mediated translational arrest does not involve a swinging of ribosomal protein L22, as previously proposed. Furthermore, bioinformatic analyses and simulations suggest nascent chain elements that may prevent translational arrest in various organisms. Altogether, the current study unveils atomic-detail interactions that explain the role of elements of TnaC and the ribosome essential for translational arrest.
Assuntos
Ribossomos/metabolismo , Triptofanase/genética , Animais , Camundongos , Simulação de Dinâmica Molecular , Óperon , Sinais Direcionadores de Proteínas/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Triptofanase/metabolismoRESUMO
The electron transfer catalyzed (ETC) repair of the DNA photolesion cyclobutane pyrimidine dimer (CPD) is mediated by the enzyme DNA photolyase. Due to its importance as part of the cancer prevention mechanism in many organisms, but also due to its unique mechanism, this DNA photoreactivation is a topic of intense study. The progress in the application of computational methods to three aspects of the ETC repair of CPD is reviewed: (i) electronic structure calculations of the cycloreversion of the CPD radical cation and radical anion, (ii) MD simulations of the DNA photolyase and its complex to photodamaged DNA, and (iii) the structure and dynamics of photodamaged DNA. The contributions of this work to the overall understanding of the reaction and its relationship to the available experimental work are highlighted.
Assuntos
Desoxirribodipirimidina Fotoliase/metabolismo , Simulação por Computador , Dano ao DNA , Reparo do DNA , Desoxirribodipirimidina Fotoliase/química , Fotoquímica , Dímeros de Pirimidina/química , Dímeros de Pirimidina/metabolismoRESUMO
Two new artificial photolyase models that recognize pyrimidine dimers in protic and aprotic organic solvents as well as in water through a combination of charge and hydrogen-bonding interactions and use a mimic of the flavine to achieve repair through reductive photoinduced electron transfer are presented. Fluorescence and NMR titration studies show that it forms a 1:1 complex with pyrimidine dimers with binding constants of approximately 10(3) M(-1) in acetonitrile or methanol, while binding constants in water at pH 7.2 are slightly lower. Excitation of the complex with visible light leads to clean and rapid cycloreversion of the pyrimidine dimer through photoinduced electron transfer catalysis. The reaction in water is significantly faster than in organic solvents. The reaction slows down at higher conversions due to product inhibition.