Extent Of Conformational Changes Research Articles

Role of Conformational Changes in DNA Recognition in Transcription Factors having Helix-Turn-Helix Motif

DNA-binding proteins play a crucial role in various cellular processes, some of these have a unique structural arrangement, the helix-turn-helix motif, which enables them to bind to DNA molecules in a specific manner. Upon binding to DNA, conformational changes occur in both the protein structure and the DNA molecule, making it essential to quantify and characterise these changes. This work aims to quantify and characterise the conformational changes in the protein structures. This is achieved by calculating the root-mean-square deviation of the protein structure in the free state and DNA bound state. A database is curated which has a significant number of DNA-bound and unbound pairs available. For the unbound structure of the proteins, pre-processing is required to remove chains other than those found in its corresponding DNA-bound structure followed by superimposing it to the DNA-bound structures obtained from the Protein Data Bank (PDB) to calculate the RMSD. After quantifying the conformational changes using RMSD values, the proteins are categorised into six types based on their observed conformational changes. In this study, the primary objective is to conduct a comprehensive analysis of conformational changes in DNA-binding proteins and investigate their correlation with various protein properties. By exploring these correlations, the study aims to unravel potential patterns or dependencies between the extent of conformational changes and the diverse properties of DNA-binding proteins. Such insights can shed light on the functional implications of structural variations in DNA-protein interactions, providing valuable information about how protein dynamics contribute to DNA recognition and binding. In addition, this work endeavours to broaden its scope by attempting to replicate DNA-bound complexes through the utilisation of freely available structures. By doing so, it aims to assess the reliability and accuracy of docking methods in predicting and reconstructing these complexes. The primary objective is to evaluate the feasibility and effectiveness of such techniques in the context of complex formation between DNA and protein molecules.

Using modern approaches to sedimentation velocity to detect conformational changes in proteins

It has been known for decades that proteins undergo conformational changes in response to binding ligands. Such changes are usually accompanied by a loss of entropy by the protein, and thus conformational changes are integral to the thermodynamics of ligand association. Methods to detect these alterations are numerous; here, we focus on the sedimentation velocity (SV) mode of AUC, which has several advantages, including ease of use and rigorous data-selection criteria. In SV, it is assumed that conformational changes manifest primarily as differences in the sedimentation coefficient (the s-value). Two methods of determining s-value differences were assessed. The first method used the widely adopted c(s) distribution to gather statistics on the s-value differences to determine whether the observed changes were reliable. In the second method, a decades-old technique called "difference SV" was revived and updated to address its viability in this era of modern instrumentation. Both methods worked well to determine the extent of conformational changes to three model systems. Both simulations and experiments were used to explore the strengths and limitations of the methods. Finally, software incorporating these methodologies was produced.

Mechanistic Insights on Ras Superfamily GTPase Switching: A Wavelet-Based Approach

Ras superfamily of small GTPases are molecular switches which regulate critical cellular processes. Three decades of structural and computational studies on this G protein family have tried to establish a general framework on their switching mechanism. The current understanding of the mechanism is that the two loops, Switch I and Switch II, undergo conformational changes upon GTP binding and hydrolysis and this change has been attributed to their functional state. However, because of variation in the extent of conformational changes seen across the members of the Ras superfamily, there is no well-defined generic modus operandi of their switching mechanism in terms of their loop conformation. Here, we have developed a novel method employing wavelet transformation to dissect the structures of these molecular switches to explore structural indices that define a unified principle of working. Our analysis shows that unique structural coupling between the P-loop, Switch I and Switch II regions, manifested in terms of wavelet coherence vectors, could act as a functional identity of the GTPases. The coupling is conserved across the entire Ras superfamily and the aberrant functioning of their oncogenic mutants is due to the disruption of this intrinsic coupling. Although similar observations were made for Ras GTPases using MD simulations, there was no structural parameter to define the coupling, as delineated here. Furthermore, the technique reported here is computationally inexpensive and can provide significant functional insights on the GTPases with as few as two structures.

Light sensitivity of the photoreceptor cryptochrome of the Drosophila circadian clock and its interaction with other clock components

Flavoprotein cryptochromes (CRYs) are blue light sensors that regulate the circadian clock in plants and animals. They consist of a highly conserved photolyase homology region (PHR) and a C‐terminal tail extension (CTE) of various sizes. In Drosophila CRY, photoreduction of the PHR bound cofactor FAD triggers conformational changes within CRY, which include undocking of C‐terminal Tail (CTT) helix. The mechanism of FAD photoreduction remains a key question in understanding CRY activation.Four tryptophan residues (W420, W397, W342, W394) mediate electron transfer for flavin photoreduction. We have substituted these residues to both redox‐inert phenylalanine residues (F) and redox‐active tyrosine (Y) residues to tune the light sensitivity of CRY. Light‐sensitivities of the variants correlate well with the extent of conformational change at the CTT. Interestingly, the surface tryptophan W394 is the most indispensable residue because it gates electron transfer from exogenous reductant to the photoreduction chain.In flies, CRY interacts with other signaling proteins of the circadian clock in a light depend‐manner to entrain the 24 hour‐cycle to environmental light. For example, in light CRY binds the protein timeless (TIM) and recruits the E3 ligase jetlag to facilitate TIM degradation. However, the molecular interactions between CRY and TIM are not well understood due to the low stability of this protein complex. We have utilized a chemical cross‐linking strategy to map the interfacing domain in this assembly and stabilize it for future structure study by structural methodsSupport or Funding InformationThis work was supported by grants from the NIH: R01GM054339 (to M.W.Y.) and R35GM122535 (to B.R.C.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Mass Spectrometry-Based Structural Assay for Activation-Dependent Conformational Changes in β-Arrestins.

β-Arrestins are key regulation proteins for G protein-coupled receptors (GPCRs) signaling. Experimental evidence suggests that β-arrestins undergo conformational changes concomitant with binding to activated, phosphorylated GPCRs. We developed a mass spectrometry-based structural proteomic assay to monitor conformational changes associated with the activation of β-arrestins. This assay utilizes synthesized phosphopeptides mimicking phosphorylated C-terminal tails of GPCRs to activate β-arrestins. The activation-dependent conformational changes of β-arrestins are revealed using limited proteolysis coupled with both SDS-PAGE and mass spectrometry analysis. As an in vitro β-arrestin activation assay, this mass spectrometry-based structural method can be adapted as a simple but useful tool to study the nature and extent of conformational changes of β-arrestins downstream of different receptors as well as β-arrestin conformations associated with different functions, such as desensitization, internalization, and signaling.

Conformational changes in antibody Fab fragments upon binding and their consequences on the performance of docking algorithms

BackgroundThe existence of conformational changes in antibodies upon binding has been previously established. However, existing analyses focus on individual cases and no quantitative study provides a more global view of potential moves and repacking, especially on recent data. The present study focuses on analyzing the conformational changes in various antibodies upon binding, providing quantitative observations to be exploited for antibody-related modeling. MethodsCartesian and dihedral Root-Mean-Squared Deviations were calculated for different subparts of 27 different antibodies, for which X-ray structures in the bound and unbound states are available. Elbow angle variations were also calculated. Previously reported results of four docking algorithms were condensed into one score giving overall docking success for each of 16 antibody-antigen cases. ResultsVery diverse movements are observed upon binding. While many loops stay very rigid, several others display side-chain repacking or backbone rearrangements, or both, at many different levels. Large conformational changes restricted to one or more antibody hypervariable loops were found to be a better indicator of docking difficulty than overall conformational variation at the antibody-antigen interface. However, the failure of docking algorithms on some almost-rigid cases shows that scoring is still a major bottleneck in docking pose prediction. ConclusionsThis study is aimed to help scientists working on antibody analysis and design by giving insights into the nature and the extent of conformational changes at different levels upon antigen binding.

Local Interaction Signal Analysis Predicts Protein-Protein Binding Affinity

Several models estimating the strength of the interaction between proteins in a complex have been proposed. By exploring the geometry of contact distribution at protein-protein interfaces, we provide animproved model of binding energy. Local interaction signal analysis (LISA) is a radial function based onterms describing favorable and non-favorable contacts obtained by density functional theory, thesupport-core-rim interface residue distribution, non-interacting charged residues and secondary structures contribution. The three-dimensional organization of the contacts and their contribution on localized hot-sites over the entire interaction surfacewere numerically evaluated. LISA achieves a correlation of 0.81 (and a root-mean-square error of 2.35± 0.38kcal/mol) when tested on 125 complexes for which experimental measurements were realized. LISA's performance is stable for subsets defined by functional composition and extent of conformational changes upon complex formation. A large-scale comparison with 17 other functions demonstrated the power of the geometrical model in the understanding of complex binding.

Chromophore-Removal-Induced Conformational Change in Photoactive Yellow Protein Determined through Spectroscopic and X-ray Solution Scattering Studies.

Photoactive yellow protein (PYP) induces negative phototaxis in Halorhodospira halophila via photoactivation triggered by light-mediated chromophore isomerization. Chromophore isomerization proceeds via a volume-conserving isomerization mechanism due to the hydrogen-bond network and steric constraints inside the protein, and causes significant conformational changes accompanied by N-terminal protrusion. However, it is unclear how the structural change of the chromophore affects the remote N-terminal domain. To understand photocycle-related structural changes, we investigated the structural aspect of chromophore removal in PYP because it possesses a disrupted hydrogen-bond network similar to that in photocycle intermediates. A comparison of the structural aspects with those observed in the photocycle would give a clue related to the structural change mechanism in the photocycle. Chromophore removal effects were assessed via UV-vis spectroscopy, circular dichroism, and X-ray solution scattering. Molecular shape reconstruction and an experiment-restrained rigid-body molecular dynamics simulation based on the scattering data were performed to determine protein shape, size, and conformational changes upon PYP bleaching. Data show that chromophore removal disrupted the holo-PYP structure, resulting in a small N-terminal protrusion, but the extent of conformational changes was markedly less than those in the photocycle. This indicates that disruption of the hydrogen-bond network alone in bleached PYP does not induce the large conformational change observed in the photocycle, which thus must result from the organized structural transition around the chromophore triggered by chromophore photoisomerization along with disruption of the hydrogen-bond network between the chromophore and the PYP core.

Predicting protein conformational changes for unbound and homology docking: learning from intrinsic and induced flexibility.

Predicting protein conformational changes from unbound structures or even homology models to bound structures remains a critical challenge for protein docking. Here we present a study directly addressing the challenge by reducing the dimensionality and narrowing the range of the corresponding conformational space. The study builds on cNMA-our new framework of partner- and contact-specific normal mode analysis that exploits encounter complexes and considers both intrinsic and induced flexibility. First, we established over a CAPRI (Critical Assessment of PRedicted Interactions) target set that the direction of conformational changes from unbound structures and homology models can be reproduced to a great extent by a small set of cNMA modes. In particular, homology-to-bound interface root-mean-square deviation (iRMSD) can be reduced by 40% on average with the slowest 30 modes. Second, we developed novel and interpretable features from cNMA and used various machine learning approaches to predict the extent of conformational changes. The models learned from a set of unbound-to-bound conformational changes could predict the actual extent of iRMSD with errors around 0.6 Å for unbound proteins in a held-out benchmark subset, around 0.8 Å for unbound proteins in the CAPRI set, and around 1 Å even for homology models in the CAPRI set. Our results shed new insights into origins of conformational differences between homology models and bound structures and provide new support for the low-dimensionality of conformational adjustment during protein associations. The results also provide new tools for ensemble generation and conformational sampling in unbound and homology docking. Proteins 2017; 85:544-556. © 2016 Wiley Periodicals, Inc.

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding.

Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca2+ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca2+-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.

Structural and Biochemical Analysis of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from the Opportunistic Pathogen Pseudomonas aeruginosa PAO1.

Biofilms are important for cell communication and growth in most bacteria, and are responsible for a number of human clinical infections and diseases. TpbA (PA3885) is a dual specific tyrosine phosphatase (DUSP) that negatively regulates biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa PAO1 by converting extracellular quorum sensing signals into internal gene cascade reactions that result in reduced biofilm formation. We have determined the three-dimensional crystal structure of wild-type TpbA from P. aeruginosa PAO1 in the phosphate-bound state and a TpbA (C132S) mutant with phosphotyrosine. Comparison between the phosphate-bound structure and the previously reported ligand-free TpbA structure reveals the extent of conformational changes that occur upon substrate binding. The largest changes occur in the functional loops that define the substrate binding site, including the PTP, general acid and α4-α5 loops. We further show that TpbA efficiently catalyzes the hydrolysis of two phosphotyrosine peptides derived from the periplasmic domain of TpbB (YfiN, PA1120), with a strong preference for dephosphorylating Tyr48 over Tyr62. This work adds to the small repertoire of DUSP structures in both the ligand-free and ligand-bound states, and provides a starting point for further study of the role of TpbA in biofilm formation.

RGD Labeled Ru(II) Polypyridyl Conjugates for Platelet Integrin αIIbβ3 Recognition and as Reporters of Integrin Conformation

The ability of two novel ruthenium(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin αIIbβ3 in solution and in live cells was described. The compounds are [Ru(bpy)2PIC-RGD](2+), bpy-RGD, and [Ru(dpp)2PIC-RGD](2+), dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2'-bipyridine, and PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified αIIbβ3 in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (Kd) in the micromolar range for each complex. The Kd for each conjugate/αIIbβ3 assembly were compared following treatment of the integrin with the activating agents, Mn(2+) and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/αIIbβ3 Kd showed relatively little variation with integrin activation, presenting the following trend: denatured αIIbβ3 > resting αIIbβ3 = pretreated DTT = pretreated Mn(2+). Kd for dpp-RGD/ αIIbβ3 showed greater variation with integrin activation and the following trend was followed: denatured αIIbβ3 > resting αIIbβ3 > pretreated Mn(2+) = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 ± 0.03 μs and 2.58 ± 0.04 μs, respectively, when the complexes were bound to resting integrin. Addition of Mn(2+) to bpy-RGD/αIIbβ3 or dpp-RGD/αIIbβ3 reduced the rotational correlation time of the ruthenium center to 1.29 ± 0.03 μs and to 1.72 ± 0.03 μs, respectively. Following treatment, the rotational correlation time decreased to 1.04 ± 0.01 μs and 1.29 ± 0.03 μs for bpy-RGD/αIIbβ3, and dpp-RGD/αIIbβ3, respectively. The large relative changes in rotational correlation times observed for Mn(2+) or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of αIIbβ3. In each case the probe colocalized with αIIb specific SZ22 antibody. Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells.

Ligand-dependent Conformational Changes in the Clamp Region of the Cardiac Ryanodine Receptor

Global conformational changes in the three-dimensional structure of the Ca(2+) release channel/ryanodine receptor (RyR) occur upon ligand activation. A number of ligands are able to activate the RyR channel, but whether these structurally diverse ligands induce the same or different conformational changes in the channel is largely unknown. Here we constructed a fluorescence resonance energy transfer (FRET)-based probe by inserting a CFP after residue Ser-2367 and a YFP after residue Tyr-2801 in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (RyR2(Ser-2367-CFP/Tyr-2801-YFP)). Both of these insertion sites have previously been mapped to the "clamp" region in the four corners of the square-shaped cytoplasmic assembly of the three-dimensional structure of RyR2. Using this novel FRET probe, we monitored the extent of conformational changes in the clamp region of RyR2(Ser-2367-CFP/Tyr-2801-YFP) induced by various ligands. We also monitored the extent of Ca(2+) release induced by the same ligands in HEK293 cells expressing RyR2(Ser-2367-CFP/Tyr-2801-YFP). We detected conformational changes in the clamp region for the ligands caffeine, aminophylline, theophylline, ATP, and ryanodine but not for Ca(2+) or 4-chloro-m-cresol, although they all induced Ca(2+) release. Interestingly, caffeine is able to induce further conformational changes in the clamp region of the ryanodine-modified channel, suggesting that ryanodine does not lock RyR in a fixed conformation. Our data demonstrate that conformational changes in the clamp region of RyR are ligand-dependent and suggest the existence of multiple ligand dependent RyR activation mechanisms associated with distinct conformational changes.

Prediction of protein–protein binding free energies

We present an energy function for predicting binding free energies of protein-protein complexes, using the three-dimensional structures of the complex and unbound proteins as input. Our function is a linear combination of nine terms and achieves a correlation coefficient of 0.63 with experimental measurements when tested on a benchmark of 144 complexes using leave-one-out cross validation. Although we systematically tested both atomic and residue-based scoring functions, the selected function is dominated by residue-based terms. Our function is stable for subsets of the benchmark stratified by experimental pH and extent of conformational change upon complex formation, with correlation coefficients ranging from 0.61 to 0.66.

Improvement of heat-induced fibril assembly of soy β-conglycinin (7S Globulins) at pH 2.0 through electrostatic screening

Protein fibril assembly at acidic conditions has recently attracted increasing interests in the fields of biochemical and food science, however, the obtained knowledge about the assembly of the proteins with complex structure (e.g. plant 7S/11S globulins) is still limited. This study investigated the influence of NaCl addition (0–300mM) on the fibril assembly of soy 7S globulins (β-conglycinin), induced by heating at 80°C and pH 2.0, with the aim of confirming the improvement of their fibril assembly by electrostatic screening. The formed fibril formation was investigated by using Th T fluorescence and atomic force microscopy (AFM) techniques. Heat-induced structure changes were traced by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism spectroscopy (CD). The results indicated that the whole fibril assembly experienced protein dissociation/hydrolysis and subsequent fibrillization process, and increasing NaCl concentration progressively increased the amount of formed fibrils, and even fibril length. AFM analyses confirmed that the increase in NaCl concentration shortened the time at which the short fibrils were visually observed, and the morphology of formed fibrils, including height of fibrils, width at half-height, and coil periodicity, slightly varied with the added NaCl concentration. The CD analyses indicated that increasing NaCl concentration greatly favored formation of β-type secondary structure, as well as extensive disruption of tertiary and/or quaternary conformations. These results confirmed that the electrostatic screening greatly improved the heat-induced fibril assembly process of soy β-conglycinin at pH 2.0. The improvement of fibril formation was largely attributed to increased extent of conformational changes at higher ionic strength. These findings would provide important information about the fibril assembly of plant oligomeric globulins at acidic pH that have important implications for the development of protein protein-based fibrillar gels.

Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation

According to their distinct biological functions, membrane-active peptides are generally classified as antimicrobial (AMP), cell-penetrating (CPP), or fusion peptides (FP). The former two classes are known to have some structural and physicochemical similarities, but fusogenic peptides tend to have rather different features and sequences. Nevertheless, we found that many CPPs and some AMPs exhibit a pronounced fusogenic activity, as measured by a lipid mixing assay with vesicles composed of typical eukaryotic lipids. Compared to the HIV fusion peptide (FP23) as a representative standard, all designer-made peptides showed much higher lipid-mixing activities (MSI-103, MAP, transportan, penetratin, Pep1). Native sequences, on the other hand, were less fusogenic (magainin 2, PGLa, gramicidin S), and pre-aggregated ones were inactive (alamethicin, SAP). The peptide structures were characterized by circular dichroism before and after interacting with the lipid vesicles. A striking correlation between the extent of conformational change and the respective fusion activities was found for the series of peptides investigated here. At the same time, the CD data show that lipid mixing can be triggered by any type of conformation acquired upon binding, whether α-helical, β-stranded, or other. These observations suggest that lipid vesicle fusion can simply be driven by the energy released upon membrane binding, peptide folding, and possibly further aggregation. This comparative study of AMPs, CPPs, and FPs emphasizes the multifunctional aspects of membrane-active peptides, and it suggests that the origin of a peptide (native sequence or designer-made) may be more relevant to define its functional range than any given name.

Trapping Small Caffeine in a Large GPCR Pocket

Three new X-ray structures of a thermostabilized A 2A adenosine receptor in complex with different antagonists reported by Dore et al. in this issue have advanced the understanding of molecular recognition for this receptor.

Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR‐type transcriptional regulators

Activation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apo- forms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.

Comparison of the Effectiveness of Chlorine, Ozone, and Photocatalytic Disinfection in Reducing the Risk of Antibiotic Resistance Pollution

AbstractEffectiveness of conventional chlorine and ozone disinfection on reduction of antibiotic resistance was compared with less commonly applied heterogeneous photocatalytic process. For this purpose plasmid DNA isolated from a multi-resistant Escherichia coli (E. coli) HB101 was treated in two different concentrations with the three oxidation processes. Oxidative damage on the plasmid DNA was analyzed with gel electrophoresis by comparing the extent of conformational changes in the DNA structure. The effectiveness of the applied oxidant in reducing the risk of resistance transfer was also evaluated by comparing the ability of treated plasmid DNA to transform competent cells. Chlorine did not affect plasmid DNA structure at the studied doses, while ozone and photocatalytic treatment resulted in conformational changes and the damage increased with increasing oxidant doses. Transformation experiments confirmed a similar trend. Chlorine did not affect the transformability and the cell counts of competent cells transformed with chlorine treated plasmid DNA were similar to those transformed by non-treated plasmid DNA in the control experiments.

The activated state of a sodium channel voltage sensor in a membrane environment

Direct structural insights on the fundamental mechanisms of permeation, selectivity, and gating remain unavailable for the Na(+) and Ca(2+) channel families. Here, we report the spectroscopic structural characterization of the isolated Voltage-Sensor Domain (VSD) of the prokaryotic Na(+) channel NaChBac in a lipid bilayer. Site-directed spin-labeling and EPR spectroscopy were carried out for 118 mutants covering all of the VSD. EPR environmental data were used to unambiguously assign the secondary structure elements, define membrane insertion limits, and evaluate the activated conformation of the isolated-VSD in the membrane using restrain-driven molecular dynamics simulations. The overall three-dimensional fold of the NaChBac-VSD closely mirrors those seen in KvAP, Kv1.2, Kv1.2-2.1 chimera, and MlotiK1. However, in comparison to the membrane-embedded KvAP-VSD, the structural dynamics of the NaChBac-VSD reveals a much tighter helix packing, with subtle differences in the local environment of the gating charges and their interaction with the rest of the protein. Using cell complementation assays we show that the NaChBac-VSD can provide a conduit to the transport of ions in the resting or "down" conformation, a feature consistent with our EPR water accessibility measurements in the activated or "up" conformation. These results suggest that the overall architecture of VSD's is remarkably conserved among K(+) and Na(+) channels and that pathways for gating-pore currents may be intrinsic to most voltage-sensors. Cell complementation assays also provide information about the putative location of the gating charges in the "down/resting" state and hence a glimpse of the extent of conformational changes during activation.