Comparative analysis of nucleotide translocation through protein nanopores using steered molecular dynamics and an adaptive biasing force.

The translocation of nucleotide molecules across biological and synthetic nanopores has attracted attention as a next generation technique for sequencing DNA. Computer simulations have the ability to provide atomistic-level insight into important states and processes, delivering a means to develop a fundamental understanding of the translocation event, for example, by extracting the free energy of the process. Even with current supercomputing facilities, the simulation of many-atom systems in fine detail is limited to shorter timescales than the real events they attempt to recreate. This imposes the need for enhanced simulation techniques that expand the scope of investigation in a given timeframe. There are numerous free energy calculation and translocation methodologies available, and it is by no means clear which method is best applied to a particular problem. This article explores the use of two popular free energy calculation methodologies in a nucleotide-nanopore translocation system, using the α-hemolysin nanopore. The first uses constant velocity-steered molecular dynamics (cv-SMD) in conjunction with Jarzynski's equality. The second applies an adaptive biasing force (ABF), which has not previously been applied to the nucleotide-nanpore system. The purpose of this study is to provide a comprehensive comparison of these methodologies, allowing for a detailed comparative assessment of the scientific merits, the computational cost, and the statistical quality of the data obtained from each technique. We find that the ABF method produces results that are closer to experimental measurements than those from cv-SMD, whereas the net errors are smaller for the same computational cost. © 2014 Wiley Periodicals, Inc

Molecular dynamics simulations of nucleotide translocation through α-hemolysin nanopores

The translocation of polynucleotides through transmembrane protein pores is a fundamental biological process with important medical and biotechnological relevance. The complex translocation process is influenced by a range of factors including the diameter and inner surface of the pore, the secondary structure of the polymer, and the interactions between the polymer and protein. Computer simulations are an invaluable means to investigate microscopic systems and thereby provide a unique, atomistic perspective of important states and processes. This thesis explores how two molecular dynamics methodologies can simulate the translocation of nucleotides through the nanopore α-hemolysin. In the first methodology, non-equilibrium constant velocity-steered simulations are combined with Jarzynski's identity to derive the free energy profiles for the passage of a polynucleotide molecule through the pore. In the second methodology, the free energy profiles are calculated from a biasing force which varies in response to energy barriers encountered during the simulation. Both approaches are used to explain the experimentally observed differences in translocation time through the nanopore between polyadenosine and polydeoxycytidine. In addition to polynucleotides, the study also investigates single nucleotide translocation. Together, the simulations highlight the role of molecular interactions between the nucleic acid molecules and the protein pore. In particular, we find that specific residues of the protein pore dominate the translocation. The unique data set helps assess two methodologies to simulate a system of considerable size and complexity

Principles of Small-Molecule Transport through Synthetic Nanopores

Synthetic nanopores made from DNA replicate the key biological processes of transporting molecular cargo across lipid bilayers. Understanding transport across the confined lumen of the nanopores is of fundamental interest and of relevance to their rational design for biotechnological applications. Here we reveal the transport principles of organic molecules through DNA nanopores by synergistically combining experiments and computer simulations. Using a highly parallel nanostructured platform, we synchronously measure the kinetic flux across hundreds of individual pores to obtain rate constants. The single-channel transport kinetics are close to the theoretical maximum, while selectivity is determined by the interplay of cargo charge and size, the pores' sterics and electrostatics, and the composition of the surrounding lipid bilayer. The narrow distribution of transport rates implies a high structural homogeneity of DNA nanopores. The molecular passageway through the nanopore is elucidated via coarse-grained constant-velocity steered molecular dynamics simulations. The ensemble simulations pinpoint with high resolution and statistical validity the selectivity filter within the channel lumen and determine the energetic factors governing transport. Our findings on these synthetic pores' structure-function relationship will serve to guide their rational engineering to tailor transport selectivity for cell biological research, sensing, and drug delivery

Molecular Dynamics Investigations of Structural Conversions in Transformer Proteins

Multifunctional proteins that undergo major structural changes to perform different functions are known as “Transformer Proteins”, which is a recently identified class of proteins. One such protein that shows a remarkable structural plasticity and has two distinct functions is the transcription antiterminator, RfaH. Depending on the interactions between its N-terminal domain and its C-terminal domain, the RfaH CTD exists as either an all-α-helix bundle or all-β-barrel structure. Another example of a transformer protein is the Ebola virus protein VP40 (eVP40), which exists in different conformations and oligomeric states (dimer, hexamer, and octamer), depending on the required function.I performed Molecular Dynamics (MD) computations to investigate the structural conversion of RfaH-CTD from its all-a to all-b form. I used various structural and statistical mechanics tools to identify important residues involved in controlling the conformational changes. In the full-length RfaH, the interdomain interactions were found to present the major barrier in the structural conversion of RfaH-CTD from all-a to all-b form. I mapped the energy landscape for the conformational changes by calculating the potential of mean force using the Adaptive Biasing Force and Jarzynski Equality methods. Similarly, the interdomain salt-bridges in the eVP40 protomer were found to play a critical role in domain association and plasma membrane (PM) assembly. This molecular dynamic simulation study is supported by virus like particle budding assays investigated by using live cell imaging that highlighted the important role of these saltbridges. I also investigated the plasma membrane association of the eVP40 dimer in various PM compositions and found that the eVP40 dimer readily associates with the PM containing POPS and PIP2 lipids. Also, the CTD helices were observed to be important in stabilizing the dimer-membrane complex. Coarse-grained MD simulations of the eVP40 hexamer and PM system revealed that the hexamer enhances the PIP2 lipid clustering at the lower leaflet of the PM. These results provide insight on the critical steps in the Ebola virus life cycle

Molecular Dynamics Simulation

Condensed matter systems, ranging from simple fluids and solids to complex multicomponent materials and even biological matter, are governed by well understood laws of physics, within the formal theoretical framework of quantum theory and statistical mechanics. On the relevant scales of length and time, the appropriate ‘first-principles’ description needs only the Schroedinger equation together with Gibbs averaging over the relevant statistical ensemble. However, this program cannot be carried out straightforwardly—dealing with electron correlations is still a challenge for the methods of quantum chemistry. Similarly, standard statistical mechanics makes precise explicit statements only on the properties of systems for which the many-body problem can be effectively reduced to one of independent particles or quasi-particles. [...

Mecanismo de ativação de canais iônicos dependentes de voltagem, Kv e Nav, e a interação com anestésicos gerais

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2013.O papel fundamental dos canais catiônicos dependentes de voltagem (VGCC) nos mais diversos organismos baseia-se no seu complexo mecanismo de ativação, i.e a transição entre dois estado fisiológicos funcionais desses canais: ativado/aberto (AT) e desativado/fechado (RT). Logo após a publicação da primeira estrutura cristalográfica do canal de mamífero Kv1.2 na conformação AT, alguns modelos do estado RT tem sido propostos na literatura para este canal. Para todos esses modelos, análises estruturais tem sugerido um consenso com os dados experimentais, destacando portanto a natureza inequívoca dessas estruturas RT. Tomados em conjunto, os estudos estruturais sobre o Kv1.2 são até agora o único conjunto de dados disponível, no nível das interações atômicas, para o entendimento sobre o mecanismo de ativação da superfamília VGCC. Recentemente, a estrutura cristalográfica de um canal de sódio de procarioto, dependente de voltagem (NavAb), foi resolvida numa conformação interpretada como estado pré-ativado do canal. Como um possível ancestral da superfamília dos canais de sódio e cálcio dependentes de voltagem de vertebrados, o surgimento da estrutura atomística do NavAb nos proporciona a primeira, e até então a única, estrutura de alta resolução para estender nossa compreensão sobre outros membros da superfamília VGCC. Dessa forma, de modo a contribuir com o referido tema, consideramos as estruturas AT e RT do Kv1.2, equilibradas na membrana, como guias estruturais em uma série de simulações de dinâmica molecular no intuito de investigar o processo de ativação do canal NavAb. Além de identificar a estrutura cristalográfica do NavAb como um estado intermediário dentro do caminho de ativação, nosso trabalho permitiu determinar conformações relacionadas aos estados fisiológicos funcionais estruturalmente relacionados às estruturas AT e RT. De maneira geral, os resultados suportam a ideia de um mecanismo de ativação altamente conservado ao longo de toda a superfamília de VGCC. ______________________________________________________________________________ ABSTRACTThe critical role of voltage-gated cation channels (VGCCs) relies on a complex voltage-dependent activation mechanism linking two physiologically relevant channel states, activated- open (AT) and resting-closed (RT) states. Following the early publication of the x-ray crystal structure of the mammalian Kv1.2 channel in the AT conformation, atomistic models for the RT state of the channel have been proposed. For all of these models, structural analyses demonstrated a consensual explanation of experimental data, thereby highlighting the unambiguous nature of these RT structures. Taken together, these structural studies on Kv1.2 have contributed so far with most of our atomic-level knowledge on the activation mechanism of VGCCs. More recently, the x-ray structure of a prokaryotic voltage-gated sodium channel, NavAb, was resolved in a conformation that was interpreted as representative of the pre-open state of the channel. As one of the possible ancestors of the large family of vertebrate voltage- + ++gated Na and Ca channels, the appearance of the NavAb structure has provided us with a first, and so far unique, template to extend our knowledge towards other members of the large family of VGCCs. Accordingly, in this contribution, we have considered the well-understood AT and RT structures of Kv1.2, equilibrated in a lipid bilayer, as guide structural models to drive a series of molecular dynamics (MD) simulations aimed at to study the activation process of NavAb. While identifying the reported NavAb structure as an intermediate conformation, not fully-activated, our work has enabled us to determine channel conformations likely related to the RT and AT states of the channel. Overall, the structural results support an activation mechanism highly conserved across the entire family of VGCCs

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals

Comparative analysis of nucleotide translocation through protein nanopores using steered molecular dynamics and an adaptive biasing force

The translocation of nucleotide molecules across biological and synthetic nanopores has attracted attention as a next generation technique for sequencing DNA. Computer simulations have the ability to provide atomistic-level insight into important states and processes, delivering a means to develop a fundamental understanding of the translocation event, for example, by extracting the free energy of the process. Even with current supercomputing facilities, the simulation of many-atom systems in fine detail is limited to shorter timescales than the real events they attempt to recreate. This imposes the need for enhanced simulation techniques that expand the scope of investigation in a given timeframe. There are numerous free energy calculation and translocation methodologies available, and it is by no means clear which method is best applied to a particular problem. This article explores the use of two popular free energy calculation methodologies in a nucleotide-nanopore translocation system, using the α-hemolysin nanopore. The first uses constant velocity-steered molecular dynamics (cv-SMD) in conjunction with Jarzynski's equality. The second applies an adaptive biasing force (ABF), which has not previously been applied to the nucleotide-nanpore system. The purpose of this study is to provide a comprehensive comparison of these methodologies, allowing for a detailed comparative assessment of the scientific merits, the computational cost, and the statistical quality of the data obtained from each technique. We find that the ABF method produces results that are closer to experimental measurements than those from cv-SMD, whereas the net errors are smaller for the same computational cost. © 2014 Wiley Periodicals, Inc

Non-covalent interactions in organotin(IV) derivatives of 5,7-ditertbutyl- and 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine as recognition motifs in crystalline self- assembly and their in vitro antistaphylococcal activity

Non-covalent interactions are known to play a key role in biological compounds due to their stabilization of the tertiary and quaternary structure of proteins [1]. Ligands similar to purine rings, such as triazolo pyrimidine ones, are very versatile in their interactions with metals and can act as model systems for natural bio-inorganic compounds [2]. A considerable series (twelve novel compounds are reported) of 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl- 1,2,4-triazolo[1,5-a]pyrimidine (dptp) were synthesized and investigated by FT-IR and 119Sn M\uf6ssbauer in the solid state and by 1H and 13C NMR spectroscopy, in solution [3]. The X-ray crystal and molecular structures of Et2SnCl2(dbtp)2 and Ph2SnCl2(EtOH)2(dptp)2 were described, in this latter pyrimidine molecules are not directly bound to the metal center but strictly H-bonded, through N(3), to the -OH group of the ethanol moieties. The network of hydrogen bonding and aromatic interactions involving pyrimidine and phenyl rings in both complexes drives their self-assembly. Noncovalent interactions involving aromatic rings are key processes in both chemical and biological recognition, contributing to overall complex stability and forming recognition motifs. It is noteworthy that in Ph2SnCl2(EtOH)2(dptp)2 \u3c0\u2013\u3c0 stacking interactions between pairs of antiparallel triazolopyrimidine rings mimick basepair interactions physiologically occurring in DNA (Fig.1). M\uf6ssbauer spectra suggest for Et2SnCl2(dbtp)2 a distorted octahedral structure, with C-Sn-C bond angles lower than 180\ub0. The estimated angle for Et2SnCl2(dbtp)2 is virtually identical to that determined by X-ray diffraction. Ph2SnCl2(EtOH)2(dptp)2 is characterized by an essentially linear C-Sn-C fragment according to the X-ray all-trans structure. The compounds were screened for their in vitro antibacterial activity on a group of reference staphylococcal strains susceptible or resistant to methicillin and against two reference Gramnegative pathogens [4] . We tested the biological activity of all the specimen against a group of staphylococcal reference strains (S. aureus ATCC 25923, S. aureus ATCC 29213, methicillin resistant S. aureus 43866 and S. epidermidis RP62A) along with Gram-negative pathogens (P. aeruginosa ATCC9027 and E. coli ATCC25922). Ph2SnCl2(EtOH)2(dptp)2 showed good antibacterial activity with a MIC value of 5 \u3bcg mL-1 against S. aureus ATCC29213 and also resulted active against methicillin resistant S. epidermidis RP62A