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Chemical transformation of polyaromatic hydrocarbon (PAH) molecules following different reaction strategies has always been the focus of organic synthesis. In this work, we report the synthesis of a PAH molecule, formation of which consists of an unusual C-C bond cleavage accompanied by a complex π-conjugated molecular scaffold rearrangement. We demonstrate that the complex chemical transformation is steered by concerted motion of individual Au

Biosensors based on graphene field-effect transistors have become a promising tool for detecting a broad range of analytes. However, they lack the stability and reproducibility required to step into biotechnological and biomedical applications. In this work, we use a controlled in-vacuum physical method for the covalent functionalization of graphene to construct ultrasensitive aptamer-based biosensors (aptasensors) able to detect hepatitis C virus core protein. These devices are highly specific and robust, achieving attomolar detection of the viral protein target in human blood plasma. The improved sensitivity is rationalized by theoretical calculations showing that induced polarization at the graphene interface caused by the proximity of covalently bound probe molecule modulates the charge balance at the graphene/molecule interface. This charge balance causes a net shift of the Dirac cone providing enhanced sensitivity towards the attomolar detection of proteins. Such an unexpected effect paves the way for using this kind of graphene-based platform for real-time diagnostics of different diseases.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Chemical Theory and Computation, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/10.1021/acs.jctc.9b00757, A double proton transfer reaction in a guanine-cytosine (GC) base pair has been proposed as a possible mechanism for rare tautomer (G*C*) formation and thus a source of spontaneous mutations. We analyze this system with free energy calculations based on extensive Quantum Mechanics/Molecular Mechanics simulations to properly consider the influence of the DNA biomolecular environment. We find that, although the G*C∗ rare tautomer is metastable in the gas phase, it is completely unstable in the conditions found in cells. Thus, our calculations show that a double proton reaction cannot be the source of spontaneous point mutations. We have also analyzed the intrabase H transfer reactions in guanine. Our results show that the DNA environment gives rise to a large free energy difference between the rare and canonical tautomers. These results show the key role of the DNA biological environment for the stability of the genetic code, This work was supported by Grants No. MAT2017-88258-R and MDM-2014-0377 (María de Maeztu Programme for Units of Excellence in R&D) from the Ministerio de Economía Industria y Competitividad (Spain)

The cohesin ring is a protein complex composed of four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. It is involved in chromosome segregation, DNA repair, chromatin organization and transcription regulation. Opening of the ring occurs at the ¿head¿ structure, formed of the ATPase domains of Smc1A and Smc3 and Rad21. We investigate the mechanisms of the cohesin ring opening using techniques of free molecular dynamics (MD), steered MD and quantum mechanics/molecular mechanics MD (QM/MM MD). The study allows the thorough analysis of the opening events at the atomic scale: i) ATP hydrolysis at the Smc1A site, evaluating the role of the carboxy-terminal domain of Rad21 in the process; ii) the activation of the Smc3 site potentially mediated by the movement of specific amino acids; and iii) opening of the head domains after the two ATP hydrolysis events. Our study suggests that the cohesin ring opening is triggered by a sequential activation of the ATP sites in which ATP hydrolysis at the Smc1A site induces ATPase activity at the Smc3 site. Our analysis also provides an explanation for the effect of pathogenic variants related to cohesinopathies and cancer., MINECO (contracts IPT2011-0964-900000 and SAF2011-13156-E to P.G-P and projects MAT2014-59966-R and “María de Maeztu” Programme for Units of Excellence in R&D; MDM-2014-0377, to J.O.); the Spanish Ministry of Health -ISCIII-Fondo de Investigación Sanitaria (FIS) (Ref.#PI15/00707, to F.J.R. and J.P.) and the Diputación General de Aragón (Grupo Consolidado B20; European Social Fund “Construyendo Europa desde Aragón”, to J.P.).

Single amino acid deletions in the β3-β4 hairpin loop of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been identified in heavily treated patients. The deletion of Asp-67 together with mutations T69G and K70R (Δ67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (Δ69) is rarely found in isolates containing TAMs. Here, we show that the complex Δ67/T69G/K70R enhances ATP-dependent phosphorolytic activity on primers terminated with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) both in the presence or absence of TAMs (i.e. M41L/T215Y), while Δ69 (or the complex S68G/Δ69/K70G) antagonize the effects of TAMs in ATP-mediated excision. These effects are consistent with AZT susceptibility data obtained with recombinant HIV-1 bearing the relevant RTs. Molecular dynamics studies based on models of wild-type HIV-1 RT and mutant Δ69, Δ67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In Δ69 RT, the side chain of Lys-70 locates away from the putative pyrophosphate binding site. Therefore, its participation in interactions required for the excision reaction is unlikely. Our theoretical studies also suggest a role for Lys-219 in thymidine analog excision/discrimination. However, pre-steady-state kinetics revealed only minor differences in selectivity of AZT-triphosphate versus dTTP between deletion-containing RTs and their homologous enzymes having the K219E mutation. K219E reduced both ATP- and pyrophosphate-mediated excision of primers terminated with thymidine analogues, only when introduced in RTs bearing Δ69 or S68G/Δ69/K70G, providing further biochemical evidence that explains the lack of association of Δ69 and TAMs in HIV-1 isolates.

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.

Bacterial cell division occurs through the formation of a protein ring (division ring) at the site of division, with FtsZ being its main component in most bacteria. FtsZ is the prokaryotic ortholog of eukaryotic tubulin; it shares GTPase activity properties and the ability to polymerize in vitro. To study the mechanism of action of FtsZ, we used molecular dynamics simulations of the behavior of the FtsZ dimer in the presence of GTP–Mg2+ and monovalent cations. The presence of a K+ ion at the GTP binding site allows the positioning of one water molecule that interacts with catalytic residues Asp235 and Asp238, which are also involved in the coordination sphere of K+. This arrangement might favor dimer stability and GTP hydrolysis. Contrary to this, Na+ destabilizes the dimer and does not allow the positioning of the catalytic water molecule. Protonation of the GTP gamma-phosphate, simulating low pH, excludes both monovalent cations and the catalytic water molecule from the GTP binding site and stabilizes the dimer. These molecular dynamics predictions were contrasted experimentally by analyzing the GTPase and polymerization activities of purified Methanococcus jannaschii and Escherichia coli FtsZ proteins in vitro., This study was supported by the Spanish Ministerio de Educación y Ciencia through grants SAF2007-61926 (to P.G.-P.) and BIO2005-02194 (to M.V.); by CIBER Fisiopatología de la Obesidad y Nutrición, an initiative of Instituto de Salud Carlos III (to P.G.-P.); by the Comunidad de Madrid through grant S-BIO-0260/2006-COMBACT (to P. G-P., J. Mingorance, and M.V.); and by the European Union through grant FP7-223431 (EU “Divinocell” project; to P.G.-P. and M.V.). Financial support from the “Fundación Ramón Areces” to CBMSO is also acknowledged. J. Mingorance was a recipient of a Ramón y Cajal fellowship financed by the European Social Fund and the Ministerio de Educación y Ciencia. A.I.R. was a recipient of an I3P contract from the Spanish Council for Scientific Research.We also thank “Centro de Cálculo Científico” (CCCUAM) for computational support. Work at Biomol- Informatics SL was partially financed by the European Social Fund

Deletions in the beta 3-beta 4 hairpin loop of human immunodeficiency virus type 1 reverse transcriptase (RT) are associated with the emergence of multidrug resistance. Common mutational patterns involve the deletion of Asp67 (Delta 67) and mutations such as K70R and T215F or T215Y, or the deletion of Thr69 (Delta 69) and mutations of the Q151M complex. Human immunodeficiency virus type 1 clones containing Delta 69 in a multidrug-resistant sequence background, including the Q151M complex and substitutions K103N, Y181C, M184V, and G190A, showed high-level resistance to all tested nucleoside RT inhibitors. In a multidrug-resistant sequence context, the deletion increases viral replication capacity. By itself, Delta 69 conferred increased susceptibility to beta-d-(+)-3'-azido-3'-deoxythymidine (AZT) and beta-l-(-)-2',3'-dideoxy-3'-thiacytidine resistance. Here, we use transient kinetics to show that, in a wild-type sequence background, Delta 69 does not affect the discrimination between AZT triphosphate and 2'-deoxythymidine 5'-triphosphate, but decreases the catalytic efficiency of the incorporation of beta-l-(-)-2',3'-dideoxy-3'-thiacytidine triphosphate relative to 2'-deoxycytidine 5'-triphosphate. In comparison with the wild-type RT, the Delta 69 mutant showed decreased ability to excise primers terminated with AZT monophosphate in the presence of ATP or pyrophosphate (PPi). These data support the role of the excision mechanism in mediating AZT hypersusceptibility. In addition, we demonstrate that the deletion has no effect on resistance to foscarnet (a PPi analogue) on phenotypic and nucleotide incorporation assays carried out with viral clones and recombinant enzymes, respectively. The results of molecular modeling studies suggest that the side chains of Lys65, Asp67, and Lys219 could play an important role in AZT hypersusceptibility mediated by Delta 69, whereas in the absence of Thr69, local structural rearrangements affecting the beta 3-beta 4 and beta 11a-beta 12 loops of the 66-kDa subunit of the RT could reduce the accessibility of the PPi donor to the terminating nucleotide at the 3' end of the primer.

The absorption of ultraviolet radiation by DNA may result in harmful genetic lesions that affect DNA replication and transcription, ultimately causing mutations, cancer, and/or cell death. We analyze the most abundant photochemical reaction in DNA, the cyclobutane thymine dimer, using hybrid quantum mechanics/molecular mechanics (QM/MM) techniques and QM/MM nonadiabatic molecular dynamics. We find that, due to its double helix structure, DNA presents a free energy barrier between nonreactive and reactive conformations leading to the photolesion. Moreover, our nonadiabatic simulations show that most of the photoexcited reactive conformations return to standard B-DNA conformations after an ultrafast nonradiative decay to the ground state. This work highlights the importance of dynamical effects (free energy, excited-state dynamics) for the study of photochemical reactions in biological systems. pre-print 512 KB

Quantum mechanics/molecular mechanics (QM/MM) methods are excellent tools for the modeling of biomolecular reactions. Recently, we have implemented a new QM/MM method (Fireball/Amber), which combines an efficient density functional theory method (Fireball) and a well-recognized molecular dynamics package (Amber), offering an excellent balance between accuracy and sampling capabilities. Here, we present a detailed explanation of the Fireball method and Fireball/Amber implementation. We also discuss how this tool can be used to analyze reactions in biomolecules using steered molecular dynamics simulations. The potential of this approach is shown by the analysis of a reaction catalyzed by the enzyme triose-phosphate isomerase (TIM). The conformational space and energetic landscape for this reaction are analyzed without a priori assumptions about the protonation states of the different residues during the reaction. The results offer a detailed description of the reaction and reveal some new features of the catalytic mechanism. In particular, we find a new reaction mechanism that is characterized by the intramolecular proton transfer from O1 to O2 and the simultaneous proton transfer from Glu 165 to C2.

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.

Structural data suggest that important hinge-bending motions of the two lobes that shape the catalytic domain of Src tyrosine kinase, together with reorganization of an alpha helix (helix C), are needed for the activation loop to adopt the catalytically competent conformation. The phosphorylation of a Tyr residue (Tyr-416) in this loop also seems to be essential for enzyme activation. However, no information is available about the dynamics of this activation process. By comparing the inactive and active forms of the catalytic domains of Src and Lck, another member of the Src family, we first identified a short stretch that can act as a hinge for the interlobe motion. The opening of the lobes was then simulated using a targeted molecular dynamics approach. The results obtained suggested that pulling the two lobes apart is not enough to induce the required conformational change in the activation loop. Rather unexpectedly, however, swinging of the lobes situated Tyr-416 in a suitable position for intramolecular autophosphorylation, and further simulation of Tyr-416-phosphorylated Src in the presence of ADP did then result in a conformational change that placed the activation loop in a position similar to that found in the active open conformation of Lck. Taken together, our results establish a physical link between intramolecular autophosphorylation and loop activation.

Quantum mechanics/molecular mechanics (QM/MM) methods are excellent tools for the modeling of biomolecular reactions. Recently, we have implemented a new QM/MM method (Fireball/Amber), which combines an efficient density functional theory method (Fireball) and a well-recognized molecular dynamics package (Amber), offering an excellent balance between accuracy and sampling capabilities. Here, we present a detailed explanation of the Fireball method and Fireball/Amber implementation. We also discuss how this tool can be used to analyze reactions in biomolecules using steered molecular dynamics simulations. The potential of this approach is shown by the analysis of a reaction catalyzed by the enzyme triose-phosphate isomerase (TIM). The conformational space and energetic landscape for this reaction are analyzed without a priori assumptions about the protonation states of the different residues during the reaction. The results offer a detailed description of the reaction and reveal some new features of the catalytic mechanism. In particular, we find a new reaction mechanism that is characterized by the intramolecular proton transfer from O1 to O2 and the simultaneous proton transfer from Glu 165 to C2.

The antitumor ecteinascidin ET743 has been shown to inhibit the transcriptional activation of a number of genes at nanomolar concentrations. Cell sensitivity to subnanomolar concentrations of the drug has also been shown to specifically depend on the transcription-coupled nucleotide excision repair system. ET743 is known to bind covalently to the minor groove of a DNA double helix in regions comprising selected sets of three consecutive base pairs. Following alkylation of a central guanine, the minor groove is widened and the DNA is bent toward the major groove. We have previously shown that in the resulting adduct the DNA triplet containing the covalently modified guanine bears a strong resemblance to a DNA triplet recognized by a C(2)H(2) zinc finger. We now expand this earlier finding and use simulation methods to show that head-to-tail binding of three ET743 molecules to three adjacent optimal binding sites stabilizes a DNA structure whose conformation is intermediate between A- and B-form DNA. Furthermore, despite the increase in roll at the sites of covalent attachment, no net curvature is apparent in this complex due to cancellation of the localized bends over virtually one turn of the helix. Both observations are in good analogy to findings in zinc finger-DNA complexes. Triplets are virtually superimposable both directly and upon shifting the register one base pair. In this latter case, the central guanine in a triplet alkylated by ET743 corresponds to the third nucleic base in the triplet recognized by a zinc finger of transcription factors such as EGR1 or Sp-1. The DNA conformation found in the ET743-DNA complex is also strongly reminiscent of an RNA-DNA hybrid, as found in the RNA polymerase II elongation complex. The possible biological implications of these findings in relation to the antitumor action of ET743 are discussed.

The complexation of the hexapeptide Lys-Cys-Thr-Cys-Cys-Ala (FT), peptidic fragment {56–61} of mouse liver metallothionein MT I, in the presence of both Cd(II) and Zn(II) is investigated as a model to understand the metal-binding properties of natural MT's, in which Cd and Zn are simultaneously present. The influence of Cd(II) or Zn(II) on the complexing behavior of FT with Zn(II) or Cd(II), respectively, is studied using the combined techniques of differential pulse polarography and multivariate curve resolution with alternating least square optimization (MCR-ALS). MCR-ALS allows both the unitary voltammograms for each electrochemical process and the concentration profiles of the different species present in the system to be obtained during the experiment, thereby providing a rough picture of the processes involved. Our results allow us to propose the formation of Cd2ZnFT2 and CdZn2FT2 complexes.

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.

Multivariate curve resolution is applied to cyclic voltammetry (CV) data obtained in the study of Cd(II) complexation by glutathione (GSH). The relatively poor resolution of CV hinders the direct interpretation of raw data. The use of CV is improved by the combination of several chemometric techniques based on factor analysis: singular-value decomposition, evolving factor analysis, and multivariate curve resolution with constrained alternating least squares optimization including a new peak-shape constraint. This multivariate analysis data treatment simultaneously reveals the formation of Cd(GSH)2 and Cd2(GSH)2 complexes, through the calculated concentration profiles, and allows the assignment of numerically obtained pure individual CV signals for the different Cd(II) ions present in solution (free and bound) and involved in the electrochemical process.

The cadmium-binding properties of the C-terminal hexapeptide of mouse metallothionein I, Lys-Cys-Thr-Cys-Cys-Ala, were studied by circular dichroism spectroscopy (CD), differential pulse polarography (DPP) and 113 Cd-nuclear magnetic resonance (NMR). The structure of the multiple cadmium binding sites could not be determined by 113 Cd-NMR because of the insolubility of the Cd–peptide samples at the high concentrations required for NMR. Therefore, alternative approaches were used: CD and DPP. The data were analyzed using a multivariate curve resolution (MCR) approach, based on factor analysis techniques, which allows the identification of the signal corresponding to different metal ions bound in different chemical environments. The CD study confirmed that the binding of Cd 2+ induces important conformational changes in the structure of the peptidic complex, including the formation of a binuclear cluster. The DPP results obtained at various Cd 2+ -to-peptide concentration ratios and pH values, under conditions where electrode adsorption is low, if not negligible, indicated the formation of different Cd 2+ –peptide complexes, and a scheme for the electrochemical reduction of the complexed Cd 2+ ions is proposed. These results show that the application of MCR to complex data, such as those from DPP, allows to reach valuable information which is not possible to be obtained by univariate approaches.

The zinc-binding properties of the C-terminal hexapeptide of mouse metallothionein I, Lys–Cys–Thr–Cys–Cys–Ala, were studied by differential pulse polarography (DPP) with a recently proposed multivariate curve resolution (MCR) method, based on factor analysis techniques, which allows the identification of the signal corresponding to different metal ions bound in different chemical environments. From DPP results obtained at different Zn 2+ -to-peptide concentration ratios and at different pH values, the formation of different Zn 2+ –peptide complexes is deduced, and a scheme for the electrochemical reduction of the complexed Zn 2+ ions is proposed. Two complexes have been detected: the first one is formed at low Zn-to-FT concentration ratios and shows a 1:2 Zn:FT stoichiometry; at higher ratios a second Zn 2+ is incorporated yielding a Zn 2 FT 2 complex which probably contains a binuclear cluster.

The complexation between Zn 2+ and glutathione (GSH), in borate buffer, is studied by Differential Pulse Polarography (DPP). Data obtained at pH 8.5 from a titration of Zn 2+ by GSH, i.e. at different GSH-to-Zn 2+ concentration ratios have been analyzed by a recently proposed multivariate curve resolution method. DPP signals obtained within a wide pH range, but at fixed 1.25:1 GSH-to-Zn 2+ concentration ratios, have been resolved individually at each pH value. Results from both sets of experiments can be explained with a complexation model involving the formation of a predominant complex, with a stoichiometry of 2:2 GSH : Zn 2+ .This result is in contrast with those for the Cd 2+ –GSH systems, where two complexes, 2:1 and 2:2 GSH : Cd 2+ , were detected. A scheme for the global electrode process is given.

Androgens are steroid hormones with a wide variety of biological actions. Biochemically, testosterone the main hormone is reduced at C5 yielding 5α and 5β-reduced androgens. Frequently, those 5-reduced androgens show biological actions different to testosterone. Besides, these compounds have either carbonyl and/or hydroxyl groups placed at carbons 3 and 17. The present work was aimed to explore the importance of such functional groups in 5α-androstane. Carbonyl or hydroxyl groups alone or combined at C3 and C17 were systematically included to 5α-androstane resulting a set of 16 different structures. The electronic structure of the 16 structures was studied using the Austin Model 1 (AM1) semi-empirical method. The results showed, that hydroxyl groups decreased the enthalpy of formation of 5α-androstane almost two-fold than carbonyl groups. Higher energy values for the HOMO and lower energy values for the LUMO were observed in structures bearing carbonyl groups. The 3-D picture of the HOMO and LUMO for the template (5α-androstane) as well as the mono- and di-hydroxy substituted structures were observed widespread along the molecules in a ‘sausage-like’ or ‘ribbon-like’ fashion. However mono- and di-keto-substituted structures showed that both HOMO and LUMO were located closer to the carbonyl groups. Carbonyl-substituted structures showed also the higher dipole moments and higher electrostatic charges. The lower values of enthalpy, dipole moments and electrostatic charges of hydroxylated molecules suggest a higher stability and lower reactivity of these compounds. Moreover, keto and di-keto structures which have the frontier orbitals at the neighborhood of these groups might facilitate a nucleophilic attack at the HOMO. It may be concluded that carbonyl groups increase the intermolecular forces of androstanes enhancing the probability to exert a biological action. Likewise, the metabolism of androstanes might be facilitated by the presence of carbonyl groups.

FtsZ filaments participate in bacterial cell division, but it is still not clear how their dynamic polymerization and shape exert force on the underlying membrane. We present a theoretical description of individual filaments that incorporates information from molecular dynamic simulations. The structure of the crystallized Methanococcus jannaschii FtsZ dimer was used to model a FtsZ pentamer that showed a curvature and a twist. The estimated bending and torsion angles between monomers and their fluctuations were included in the theoretical description. The MD data also permitted positioning the curvature with respect to the protein coordinates and allowed us to explore the effect of the relative orientation of the preferred curvature with respect to the surface plane. We find that maximum tension is attained when filaments are firmly attached and oriented with their curvature perpendicular to the surface and that the twist serves as a valve to release or to tighten the tension exerted by the curved filaments on the membrane. The theoretical model also shows that the presence of torsion can explain the shape distribution of short filaments observed by Atomic Force Microscopy in previously published experiments. New experiments with FtsZ covalently attached to lipid membranes show that the filament on-plane curvature depends on lipid head charge, confirming the predicted monomer orientation effects. This new model underlines the fact that the combination of the three elements, filament curvature, twist and the strength and orientation of its surface attachment, can modulate the force exerted on the membrane during cell division. © 2014 The Royal Society of Chemistry., Comunidad de Madrid; to MVe), DIVINOCELL FP7 HEALTHF3-2009-223431 (European Commision to MVe and P.G-P.), Plan Nacional BIO2008-04478-C03-00 (Ministerio de Ciencia e Innovación; to MVe), CONSOLIDER INGENIO 2010 CSD2007-00010 (Ministerio de Ciencia e Innovación; to MVe) FIS2010-22047-C05-01 (Ministerio de Ciencia e Innovación,to P.T.)

The complexation of glutathione (GSH) by Cd2+ has been investigated as a model for the coordination chemistry of Cd2+ by thiol-containing peptides. Experimental data obtained by differential pulse polarography (DPP) for different Cd2+-to-GSH concentration ratios at fixed pH have been globally analyzed by the recently proposed multivariate curve resolution (MCR) method. Polarograms from experiments at fixed Cd2+-to-GSH concentration ratios but changing pH values have been univariately deconvoluted at each pH value. In both cases, the obtained single DPP peaks can be associated to specific electrode processes. The results obtained in this work, together with structural data from the literature, allow us to propose a model for the complexation of Cd2+ by GSH involving the formation of Cd(GSH)2 and Cd2(GSH)2 complexes. Moreover, a scheme for the electrode process is also proposed.

Thymidine analogue resistance mutations (TAMs) in HIV-1 reverse transcriptase (RT) associate in two clusters: (i) TAM1 (M41L, L210W and T215Y) and TAM2 (D67N, K70R, K219E/Q, and sometimes T215F). The amino acid substitution H208Y shows increased prevalence in patients treated with nucleoside analogues and is frequently associated with TAM1 mutations. We studied the molecular mechanism favoring the selection of H208Y in the presence of zidovudine, tenofovir and other nucleoside RT inhibitors (NRTIs). NRTI susceptibility was not affected by the addition of H208Y in phenotypic assays carried out in MT-4 cells using recombinant HIV-1 containing wild-type (subtype B, BH10), H208Y, M41L/L210W/T215Y or M41L/H208Y/L210W/T215Y RTs. However, enzymatic studies carried out with purified RTs revealed that in the presence of M41L/L210W/T215Y, H208Y increases the RT’s ability to unblock and extend primers terminated with zidovudine, tenofovir and in a lesser extent, stavudine. These effects were observed with DNA/DNA complexes (but not with RNA/DNA) and resulted from the higher ATP-dependent excision activity of the M41L/H208Y/L210W/T215Y RT compared with the M41L/L210W/T215Y mutant. The increased rescue efficiency of the M41L/H208Y/L210W/T215Y RT was observed in the presence of ATP but not with GTP or ITP. Molecular dynamics studies predict an alteration of the stacking interactions between Tyr215 and the adenine ring of ATP due to long-distance effects caused by tighter packaging of Tyr208 and Trp212. These studies provide a mechanistic explanation for the association of TAM-1 and H208Y mutations in viral isolates from patients treated with NRTIs.

A systematic study on the impact of carbonyl and hydroxyl functional groups on the electronic structure of the steroid, androst-4-ene (androstene) was carried out by the semiempirical methods, AM1 and PM3. A total of 16 molecules from androstene were studied with mono and disubstituted hydroxyl and carbonyl groups at C3 and C17. Results showed the lowering of total energies and enthalpies of formation with the addition of the functional groups. HOMO was located always along of the C4C5 π-bond. The LUMO occupied the same place except for two 17-keto androstene structures which showed the LUMO at C17 and degeneracy at LUMO and LUMO + 1. Structures with 3-keto conformation were the most conspicuous showing higher dipole moment, lower energy in both HOMO and LUMO, some degeneracy with HOMO and HOMO-1 and more impact than the hydroxyl groups in the charges of neighbor carbon atoms. Interestingly, this conformation which conjugates O  C3C4  C5, belongs to the ring A conformation of testosterone, epitestosterone and androstenedione, three well known androgens. It is concluded that the double bond of androstenes at C4C5 when conjugated with a carbonyl group at C3 enhances the likelihood of biological activity.

The complexation of Cd2+ by glutathione (GSH), in 0.13 m borate buffer at pH 9.5, was studied by differential pulse polarography (DPP) and multivariate curve resolution. The Cd-GSH system has been chosen as a model to check the possibilities of this new polarographic approach to the study of metal ion complexation by peptides. Experimental data obtained by DPP for different Cd2+-to-GSH concentration ratios have been analyzed by a procedure which consists of using several chemometrical techniques based on factor analysis: principal component analysis, evolving factor analysis, and multivariate curve resolution with alternating least-squares (ALS) optimization. The use of different constraints during the ALS optimization process, such as nonnegativity and unimodality constraints, yields the optimal sought solution from a chemical point of view. In the present work, a new constraint has been implemented during ALS optimization to take into account the expected peak-shaped signal of DPP. This data treatment allows us to detect simultaneously the formation of 1:1 and 1:2 Cd:GSH complexes which were very difficult to detect by univariate analysis of DPP data. It is concluded that the described multivariate curve resolution method could be a reliable tool for studying metal-binding properties of peptides.

An electrochemical study, using differential pulse polarography, of the binding properties of the peptide Lys-Cys-Thr-Cys-Cys-Ala Thionein Fragment [56–61] MT I (FT) with cadmium and zinc was performed. This molecule, intrinsic to the metallothionein structure, was chosen as a model of the Cd, Zn Metallothioneins ion exchange and binding properties. The influence of the addition of metal ions, cadmium and/or zinc was studied with different ratios of complexing ligand/metallic cations, FT/M(II). It is assumed that the main complex M(II)-FT possesses a stoichiometry of 1:1, but probably two or more complexes co-exist depending on the FT/M ratios. The equilibrium M + FT ⇌ MFT concerning the complexation equilibrium of formation and dissociation was investigated as a function of pH, going from acid to basic solution and vice versa in different mixtures: Cd:FT, Zn:FT and Cd:Zn:FT. The apparent stability constants of the complexes Cd-FT, Zn-FT were estimated at different pH values.

In order to obtain a better understanding of the electrochemical behavior of the Metallothionein (MT), the peptidic fragment Lys–Cys–Thr–Cys–Cys–Ala [56–61] MT I (FT) compound constitutive of the proteic structure has been studied. The elucidation of the electrochemical mechanism at the electrode could be reached from comparison with other mechanisms proposed for similar molecules. This study stems from a more complete work. Under our experimental conditions, with a low concentration of the substance (< 5 × 10−5 M) and the use of a dropping mercury electrode, a diffusion controlled mechanism is suggested for the electrochemical behavior of FT. In this case the mercury electrode itself is involved in the charge transfer step and the polarographic response is attributed to the oxidation of the mercury electrode in the presence of the chelating agent FT. This hypothesis was supported by the lack of modification of the electrocapillary curves in the presence of low concentration of FT in relation to those obtained with the supporting electrolyte alone and by the linear relationship between the polarographic intensity and the concentration at a low concentration of FT. The influence of the pH of the solution on the behavior of FT was investigated using DC and DPP methods. The variation of i1, ip and Ep with pH was determined. From these results the apparent acid-base dissociation constant of the FT was evaluated, corresponding to the dissociation of protons from the thiol groups. Likewise the apparent stability constants of the Hg–FT were estimated as a function of the pH. It was assumed that the main complex of Hg–FT possessed a stoichiometry of 1 : 1 but it is probable that two or more different complexes coexisted.

The electrochemical behaviour of Cd,Zn thioneins (MT) from rabbit liver and horse kidney with respect to solution pH at the dropping mercury electrode was studied using differential pulse polarography. The equilibrium M + T ⇆ MT concerning the formation of metallothionein from their metal depleted form for assessing the possibility of reproducing the same original molecule was investigated as a function of pH, going from basic to acid solutions and vice versa. The equilibrium is reversible and dissociation and association are quickly reached. Consequently the apothionein, metallothionein free of metal ions and exhibiting a mainly random-coil structure, in acid solution is still able to maintain its metal binding capability and in neutral or basic solution to complex metal ions through its thiol groups. However, the polarographic response from acid to basic solution is not the same as for the variation of pH in the opposite direction. It is possible that the reconstituted Cd, Zn thionein possesses properties and characteristics which are different to their native form. The apparent stability constants of the complexes CdT, ZnT and HgT were estimated at different pH values. The order of affinity of the metal ions to the thionein is well determined: KHgT ⪢ KCdT > KZnT.

Bacteriophage Ø29 transcriptional regulator, protein p4, interacts with its DNA target employing two mechanisms: direct readout of the chemical signatures of only one DNA base, and inducing local modification on the topology of short A-tracts (indirect readout). P4 binds as a dimer to targets consisting of imperfect inverted repeats. Here, we use Molecular Dynamic simulation to define interactions of a cluster of 12 positive charged amino acids of p4 with DNA and biochemical assays with modified DNA targets and mutated proteins to quantify the contribution of residues in the nucleoprotein complex. Our results show the implication of Arg54, with base-unspecific interaction in the central A-tract, in p4 binding affinity. Despite being chemically equivalent and in identical protein monomers, the two Arg54 residues differed in their interactions with DNA. We discuss an indirect-readout mechanism for p4-DNA recognition mediated by dissimilar interaction of arginines penetrating the minor groove and the inherent properties of the A-tract. Our finding extent the current understanding of protein-DNA recognition and contributes to the relevance of the sequence-dependent conformational malleability of the DNA, enlightening the role of arginines in binding affinity. Characterization of mutant p4R54A shows that the residue is required for the activity of the protein as transcriptional regulator., This work was supported by the Ministerio de Ciencia e Innovación of Spain [Grants BFU2008-01216/BMC to A.C., BFU2008- 437 00215/BMC to M.S.]; by the Madrid Autonomous Community [Grant S-2009/MAT/1507 to M.S.]; and by an Institutional Grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa.

The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from β-sheet conformation to an elongated coil and then spontaneously to an α-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer. © 2012 Elsevier Inc., Spanish Ministerio de Ciencia e Innovación (SAF2007-61926, IPT2011-0964-900000 and SAF2011 13156-E); the Madrid regional authority through Grant S-BIO-0260/2006-COMBACT; and the European Commission through Grants FP7 HEALTH-F3-2009-223431 (EU Project ‘‘Divinocell’’) and FP7 HEALTH-2011-278603 (EU Project ‘‘Dorian); Fundación Ramón Areces

Activation of the water molecule involved in GTP hydrolysis within the HRas RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln 61 will stabilize the transient OH and H 3 O þ molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant., Ministerio de Ciencia e Innovación (SAF2007-61926); Madrid Regional Authority (S-BIO-0260/2006-COMBACT); European Commission (FP7 HEALTH-F3-2009-223431); European Social Fund, Fundación Ramón Areces

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.

FtsZ, the prokaryotic ortholog of tubulin, assembles into polymers in the bacterial division ring. The interfaces between monomers contain a GTP molecule, but the relationship between polymerization and GTPase activity is not unequivocally proven. A set of short FtsZ polymers were modelled and the formation of active GTPase structures was monitored using molecular dynamics. Only the interfaces nearest the polymer ends exhibited an adequate geometry for GTP hydrolysis. Simulated conversion of interfaces from close-to-end to internal position and vice versa resulted in their spontaneous rearrangement between active and inactive conformations. This predicted behavior of FtsZ polymer ends was supported by in vitro experiments. © 2012 Federation of European Biochemical Societies., Ministerio de Ciencia e Innovación (BIO2008-04178, SAF2007-61926, IPT2011-0964-900000, SAF2011-13156-E); Comunidad de Madrid (S-BIO-0260/2006; European Commission (FP7 HEALTH-F3-2009-223431; HEALTH-2011- 278603); Ramón y Cajal ; European Social Fund to Biomol-Informatics; Fundación Ramón Areces

Signaling networks are key elements in all major aspects of cellular life, playing a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. The latter comprises a whole range of networks that are involved in regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses. The key mechanism involves the transduction of extracellular signals across the cell surface to different effectors in the cytosol and the nucleus. Dysregulation of these pathways is often associated with immunology disorders and malignant diseases such as cancer. One of the most common mechanisms of activation and/or inactivation of signaling transduction pathways is phosphorylation and de-phosphorylation at serine, threonine and tyrosine residues. Phosphoproteomics is playing an important role in our understanding of how phosphorylation participates in translating distinct signals into the normal and or abnormal physiological responses, and has shifted research towards screening for potential therapies for diseases and in-depth analysis of phosphoproteomes. Given the importance of phosphoproteomics in translational research we aim at outlining phosphoproteomic approaches based on mass spectrometry (MS). This review focuses on (1b) the role of phospho signaling in immunology, (2a) current phosphopeptide enrichment methods based on IMAC and titanium dioxide, (2b) phosphopeptide analysis by MS, and (2c) issues concerned with interpretation of phospho spectra by database dependent search. Finally, quantitative methods used in phosphoproteomics such as Stable Isotope labeling with Amino acid in cell Culture (SILAC), isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and Absolute Quantification (AQUA) is discussed in section 3.

Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools., This study was supported by: the Spanish Ministerio de Ciencia e Innovación through grants SAF2007-61926 (to PGP) and the European Commission through grant FP7 HEALTH-F3-2009-223431 (to PGP). Biomol-Informatics was financed by the European Social Fund. Support from the "Fundación Ramón Areces" is acknowledged. We also thank the Centro de Computación Científica-UAM for computational support. Special thanks Prof. Ernest Feytmans (Honorary Director at Swiss Institute of Bioinformatics -Location Geneva Area, Switzerland) and Prof. Shabaz Mohammed (Theme Leader at the Netherlands Proteomics Centre, Lecturer Utrecht University) who contributed to the publication of this article.

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined (“canalized”) pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions (“calcium rivet”) that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely “decanalized” and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched “mechanical antenna” with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.

Polylysine has been covalently bound to human transferrin in a 1:1 molar ratio over a disulfide bond that can be easily split by reducing agents such as dithiothreitol. The association constant for the binding of the transferrin-polylysine derivative to transferrin receptors present on rat erythroblasts and the number of binding sites were identical to the corresponding values found for native transferrin. The incubation of the cells with transferrin-polylysine affected the partitioning of erythroblasts in a charge-sensitive aqueous two-phase system containing Dextran and polyethylene glycol. The polylysine part introduced a nonspecific influence on the partitioning that could be eliminated by preincubation of the cells with an excess of sialic acid. The partition ratio, G, of the erythroblasts changed with a factor of 1.9 for each set of 100,000 polylysine chains attached per cell.

The structural and dynamical changes occurring before nucleotide addition were studied using molecular dynamics (MD) simulations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexes containing one or two Mg2+ ions in the presence of dNTP. Our models revealed that the formation of a catalytically competent DNA polymerase complex required subtle rearrangements at the catalytic site A, which occurred only when an Mg2+ ion was bound. This model has been validated using pre-steady-state kinetics to show that free Mg2+ is necessary to obtain a catalytically competent polymerase. Kinetic studies carried out with Be2+ as a cofactor permitted the functional discrimination between metal sites A and B. At low concentrations, Be2+ increased the catalytic efficiency of the polymerase, while at higher concentrations, it competed with Mg2+ for binding to site A, and inhibited DNA polymerization. In agreement with experimental data, MD simulations revealed that the catalytic attack distance between the 3-OH of the primer and the phosphorus in complexes containing Be2+ instead of Mg2+ at site A was above 4.5 A. Our findings provide a detailed description of the mechanism of DNA polymerization and should be helpful to understand the molecular basis of DNA replication fidelity.

Human respiratory syncytial virus (HRSV) P protein, 241 amino acid long, is a structural homotetrameric phosphoprotein. Viral transcription and replication processes are dependent on functional P protein interactions inside viral ribonucleoprotein complexes (RNPs). Binding capacity to RNPs proteins and transcription and replication complementation analyses, using inactive P protein variants, have identified residues essential for functional interactions with itself, L, N and M2-1 proteins. P protein may establish some of these interactions as monomer, but efficient viral transcription and replication requires P protein oligomerization through the central region of the molecule. A structurally stable three-dimensional model has been generated in silico for this region (residues 98-158). Our analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization. Additionally, the residues D136, S156, T160 and E179 appear to be essential for P protein activity on viral RNA synthesis and very high turnover phosphorylation at S143, T160 and T210 could regulate it. Thus, compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect.