Supramolecular Organization of Heptapyrenotide Oligomers—An in Depth Investigation by Molecular Dynamics Simulations

We present a molecular modeling study based on ab initio and classical molecular dynamics calculations, for the investigation of the tridimensional structure and supramolecular assembly formation of heptapyrenotide oligomers in water solution. Our calculations show that free oligomers self-assemble in helical structures characterized by an inner core formed by π- stacked pyrene units, and external grooves formed by the linker moieties. The coiling of the linkers has high ordering, dominated by hydrogen-bond interactions among the phosphate and amide groups. Our models support a mechanism of longitudinal supramolecular oligomerization based on interstrand pyrene intercalation. Only a minimal number of pyrene units intercalate at one end, favoring formation of very extended longitudinal chains, as also detected by AFM experiment. Our results provide a structural explanation of the mechanism of chirality amplification in 1:1 mixtures of standard heptapyrenotides and modified oligomers with covalently linked deoxycytidine, based on selective molecular recognition and binding of the nucleotide to the groove of the left-wound helix.

Unraveling the Conformational Determinants of Peptide Dendrimers Using Molecular Dynamics Simulations

Peptide dendrimers are synthetic tree-like molecules composed of amino acids. There are at least two kinds of preferential structural behaviors exhibited by these molecules, which acquire either compact or noncompact shapes. However, the key structural determinants of such behaviors remained, until now, unstudied. Herein, we conduct a comprehensive investigation of the structural determinants of peptide dendrimers by employing long molecular dynamics simulations to characterize an extended set of third generation dendrimers. Our results clearly show that a trade-off between electrostatic effects and hydrogen bond formation controls structure acquisition in these systems. Moreover, by selectively changing the dendrimers charge we are able to manipulate the exhibited compactness. In contrast, the length of branching residues does not seem to be a major structural determinant. Our results are in accordance with the most recent experimental evidence and shed some light on the key molecular level interactions controlling structure acquisition in these systems. Thus, the results presented constitute valuable insights that can contribute to the development of truly tailor-made dendritic systems.

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

Modeling of human P450 oxidoreductase structure by in silico mutagenesis and MD simulation

P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450s and mutations in POR cause several metabolic disorders. We have modeled the structure of human P450 oxidoreductase by in silico amino acid replacements in the rat POR crystal structure. The rat POR has 94% homology with human POR and 38 amino acids were replaced to make its sequence identical to human POR. Several rounds of molecular dynamic simulations refined the model and removed structural clashes from side chain alterations of replaced amino acids. This approach has the advantage of keeping the cofactor contacts and structural features of the core enzyme intact which could not be achieved by homology based approaches. The final model from our approach was of high quality and compared well with experimentally determined structures of other PORs. This model will be used for analyzing the structural implications of mutations and polymorphisms in human POR.

Sulfamethoxazole induces a switch mechanism in T cell receptors containing TCRVβ20-1, altering pHLA recognition

T cell receptors (TCR) containing Vβ20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vβ20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2β domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vβ20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable β or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2β loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2β loop amino acid positions. These residues, TYRβ57, ASPβ64, and LYSβ65 initially hold hydrogen bonds from the CDR2β to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2β of Vβ20-1 acts as a ligand controlled switch affecting overall TCR binding affinity.

Dynamic computer simulations of electrophoresis: a versatile research and teaching tool

Software is available, which simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. These dynamic models are based upon equations derived from the transport concepts such as electromigration, diffusion, electroosmosis and imposed hydrodynamic buffer flow that are applied to user-specified initial distributions of analytes and electrolytes. They are able to predict the evolution of electrolyte systems together with associated properties such as pH and conductivity profiles and are as such the most versatile tool to explore the fundamentals of electrokinetic separations and analyses. In addition to revealing the detailed mechanisms of fundamental phenomena that occur in electrophoretic separations, dynamic simulations are useful for educational purposes. This review includes a list of current high-resolution simulators, information on how a simulation is performed, simulation examples for zone electrophoresis, ITP, IEF and EKC and a comprehensive discussion of the applications and achievements.

Single Molecular Conductance of Tolanes: Experimental and Theoretical Study on the Junction Evolution Dependent on the Anchoring Group

Employing a scanning tunneling microscopy based beak junction technique and mechanically controlled break junction experiments, we investigated tolane (diphenylacetylene)-type single molecular junctions having four different anchoring groups (SH, pyridyl (PY), NH2, and CN) at a solid/liquid interface. The combination of current–distance and current–voltage measurements and their quantitative statistical analysis revealed the following sequence for junction formation probability and stability: PY > SH > NH2 > CN. For all single molecular junctions investigated, we observed the evolution through multiple junction configurations, with a particularly well-defined binding geometry for PY. The comparison of density functional theory type model calculations and molecular dynamics simulations with the experimental results revealed structure and mechanistic details of the evolution of the different types of (single) molecular junctions upon stretching quantitatively.

Computer simulations of sample preconcentration in carrier-free systems and isoelectric focusing in microchannels using simple ampholytes.

In this work, electrophoretic preconcentration of protein and peptide samples in microchannels was studied theoretically using the 1D dynamic simulator GENTRANS, and experimentally combined with MS. In all configurations studied, the sample was uniformly distributed throughout the channel before power application, and driving electrodes were used as microchannel ends. In the first part, previously obtained experimental results from carrier-free systems are compared to simulation results, and the effects of atmospheric carbon dioxide and impurities in the sample solution are examined. Simulation provided insight into the dynamics of the transport of all components under the applied electric field and revealed the formation of a pure water zone in the channel center. In the second part, the use of an IEF procedure with simple well defined amphoteric carrier components, i.e. amino acids, for concentration and fractionation of peptides was investigated. By performing simulations a qualitative description of the analyte behavior in this system was obtained. Neurotensin and [Glu1]-Fibrinopeptide B were separated by IEF in microchannels featuring a liquid lid for simple sample handling and placement of the driving electrodes. Component distributions in the channel were detected using MALDI- and nano-ESI-MS and data were in agreement with those obtained by simulation. Dynamic simulations are demonstrated to represent an effective tool to investigate the electrophoretic behavior of all components in the microchannel.

Pi release from myosin: a simulation analysis of possible pathways

The release of phosphate (Pi) is an important element in actomyosin function and has been shown to be accelerated by the binding of myosin to actin. To provide information about the structural elements important for Pi release, possible escape pathways from various isolated myosin II structures have been determined by molecular dynamics simulations designed for studying such slow processes. The residues forming the pathways were identified and their role was evaluated by mutant simulations. Pi release is slow in the pre-powerstroke structure, an important element in preventing the powerstroke prior to actin binding, and is much more rapid for Pi modeled into the post-rigor and rigor-like structures. The previously proposed backdoor route is dominant in the pre-powerstroke and post-rigor states, whereas a different path is most important in the rigor-like state. This finding suggests a mechanism for the actin-activated acceleration of Pi release.

Block of the hERG channel by bupivacaine: Electrophysiological and modeling insights towards stereochemical optimization

The hERG voltage-gated potassium channel mediates the cardiac I(Kr) current, which is crucial for the duration of the cardiac action potential. Undesired block of the channel by certain drugs may prolong the QT interval and increase the risk of malignant ventricular arrhythmias. Although the molecular determinants of hERG block have been intensively studied, not much is known about its stereoselectivity. Levo-(S)-bupivacaine was the first drug reported to have a higher affinity to block hERG than its enantiomer. This study strives to understand the principles underlying the stereoselectivity of bupivacaine block with the help of mutagenesis analyses and molecular modeling simulations. Electrophysiological measurements of mutated hERG channels allowed for the identification of residues involved in bupivacaine binding and stereoselectivity. Docking and molecular mechanics simulations for both enantiomers of bupivacaine and terfenadine (a non-stereoselective blocker) were performed inside an open-state model of the hERG channel. The predicted binding modes enabled a clear depiction of ligand-protein interactions. Estimated binding affinities for both enantiomers were consistent with electrophysiological measurements. A similar computational procedure was applied to bupivacaine enantiomers towards two mutated hERG channels (Tyr652Ala and Phe656Ala). This study confirmed, at the molecular level, that bupivacaine stereoselectively binds the hERG channel. These results help to lay the foundation for structural guidelines to optimize the cardiotoxic profile of drug candidates in silico.

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy DeltaH. Arranging the tc-residues in a continuous fashion rescues T(m) and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T(m) when paired to complementary DNA and leads to substantial increases in T(m) when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.

Engineering Tocopherol Selectivity in alpha-TTP: A Combined In Vitro/In Silico Study

"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of a-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover," "the identification of a c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."

Factor XIII activation peptide is released into plasma upon cleavage by thrombin and shows a different structure compared to its bound form

The first step of coagulation factor XIII (FXIII) activation involves cleavage of the FXIII activation peptide (FXIII-AP) by thrombin. However, it is not known whether the FXIII-AP is released into plasma upon cleavage or remains attached to activated FXIII. The aim of the present work was to study the structure of free FXIII-AP, develop an assay for FXIII-AP determination in human plasma, and to answer the question whether FXIII-AP is released into plasma. We used ab-initio modeling and molecular dynamics simulations to study the structure of free FXIII-AP. We raised monoclonal and polyclonal antibodies against FXIII-AP and developed a highly sensitive and specific ELISA method for direct detection of FXIII-AP in human plasma. Structural analysis showed a putative different conformation of the free FXIII-AP compared to FXIII-AP bound to the FXIII protein. We concluded that it might be feasible to develop specific antibodies against the free FXIII-AP. Using our new FXIII-AP ELISA, we found high levels of FXIII-AP in in-vitro activated plasma samples and serum. We showed for the first time that FXIIIAP is detached from activated FXIII and is released into plasma, where it can be directly measured. Our findings may be of major clinical interest in regard to a possible new marker in thrombotic disease.