Conformational Motions Research Articles

Exploration of Conformational Selection During Translesion Synthesis: In Silico Studies of DinB

E. coli DNA Polymerase IV, also named DinB after its encoding gene, is a Y‐family DNA polymerase used by cells during stress‐induced mutagenesis. DinB functions as an error‐prone polymerase implicated in translesion synthesis. Kinetics studies have shown that DinB is more efficient at bypassing damaged DNA versus undamaged DNA, and that the polymerase is more effective at bypassing minor groove mutations, such as the N2‐furfuryl‐dG, than major groove mutations, such as the O6‐methylated‐dG. Recent hydrogen experiments suggest that DinB undergoes a conformational change only in the presence of select damages on the DNA template strand. These differentiations imply a possible mechanism of bypass discrimination, though discrete characteristic movements have not been identified. In this study, molecular dynamics (MD) simulations were used to explore these conformational movements through the generation and study of various systems of DNA/protein/incoming nucleotide combinations. A total of four systems were made: binary (DNA and protein), ternary‐CG (DNA, protein, correct incoming nucleotide), ternary O6‐methylated‐dG (deoxyguanosine with O6‐methylation on the template strand, protein, and correct incoming nucleotide), and binary O6‐methylated‐dG (deoxyguanosine with O6‐methylation on the template strand and protein). A ternary‐GG (DNA, protein, incorrect incoming nucleotide) system has been built, but has not yet been run through simulations. An N2‐furfuryl‐dG molecule was also built; however, the molecule requires further parameterization prior to dynamics run. The binary systems showed the highest levels of fluctuation, implying that, in lack of substrate, DinB has no conformational selectivity and is thus in a floppy and unrestrained state. Initially, the ternary O6‐methylated‐dG system seemed more similar to the ternary‐CG system than the binary system (the opposite of what was predicted). However, closer analysis of the ternary O6‐methylated‐dG system showed that residues key to stabilization of the incoming nucleotide were missing from the active site, suggesting an inability to reach catalytic competence without these residues to promote the appropriate conformational selection. Previously, it was believed that DinB adopted either an open‐inactive conformation (in the presence of a mutation or lack of substrate) or closed‐active conformation (no mutation and presence of proper substrate) in accordance with the induced‐fit theory of catalysis. However, our data suggest a less rigid breathing movement and mechanism of conformational selection as opposed to induced fit. Accordingly, data interpretation has moved from analysis of gross domain movements to analysis of the presence of key catalytic residues in the active site.Support or Funding InformationThis work was supported in part by the Rose M. Badgeley Residuary Charitable Trust Award and the Colette Mahoney Award to BSB.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Insight into microtubule destabilization mechanism of 3,4,5-trimethoxyphenyl indanone derivatives using molecular dynamics simulation and conformational modes analysis.

Colchicine site inhibitors are microtubule destabilizers having promising role in cancer therapeutics. In the current study, four such indanone derivatives (t1, t9, t14 and t17) with 3,4,5-trimethoxyphenyl fragment (ring A) and showing significant microtubule destabilization property have been explored. The interaction mechanism and conformational modes triggered by binding of these indanone derivatives and combretastatin at colchicine binding site (CBS) of αβ-tubulin dimer were studied using molecular dynamics (MD) simulation, principle component analysis and free energy landscape analysis. In the MD results, t1 showed binding similar to colchicine interacting in the deep hydrophobic core at the CBS. While t9, t14 and t17 showed binding conformation similar to combretastatin, with ring A superficially binding at the CBS. Results demonstrated that ring A played a vital role in binding via hydrophobic interactions and got anchored between the S8 and S9 sheets, H8 helix and T7 loop at the CBS. Conformational modes study revealed that twisting and bending conformational motions (as found in the apo system) were nearly absent in the ligand bound systems. Absence of twisting motion might causes loss of lateral contacts in microtubule, thus promoting microtubule destabilization. This study provides detailed account of microtubule destabilization mechanism by indanone ligands and combretastatin, and would be helpful for designing microtubule destabilizers with higher activity.

Multiscale modeling of keratin, collagen, elastin and related human diseases: Perspectives from atomistic to coarse-grained molecular dynamics simulations

Scleroproteins are an important category of proteins within the human body that adopt filamentous, elongated conformations in contrast with typical globular proteins. These include keratin, collagen, and elastin, which often serve a common mechanical function in structural support of cells and tissues. Genetic mutations alter these proteins, disrupting their functions and causing diseases. Computational characterization of these mutations has proven to be extremely valuable in identifying the intricate structure–function relationships of scleroproteins from the molecular scale up, especially if combined with multiscale experimental analysis and the synthesis of model proteins to test specific structure–function relationships. In this work, we review numerous critical diseases that are related to keratin, collagen, and elastin, and through several case studies, we propose ways of extensively utilizing multiscale modeling, from atomistic to coarse-grained molecular dynamics simulations, to uncover the molecular origins for some of these diseases and to aid in the development of novel cures and therapies. As case studies, we examine the effects of the genetic disease Epidermolytic Hyperkeratosis (EHK) on the structure and aggregation of keratins 1 and 10; we propose models to understand the diseases of Osteogenesis Imperfecta (OI) and Alport syndrome (AS) that affect the mechanical and aggregation properties of collagen; and we develop atomistic molecular dynamics and elastic network models of elastin to determine the role of mutations in diseases such as Cutis Laxa and Supravalvular Aortic Stenosis on elastin’s structure and molecular conformational motions and implications for assembly.

Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization.

Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodology relies on "painting" the reactive and solvent-exposed amino acid residues with chemical tags and using the pattern of modifications as footprints from analysis by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in solution or even in cells and is complementary to those of X-ray crystallography and NMR. It is particularly useful in mapping protein-ligand interfaces and conformational changes resulting from ligand binding, mutation, and aggregation. Fast photochemical oxidation of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that utilizes a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce solution hydroxyl radicals, which subsequently modify the protein in situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to approximately microsecond. Thus, the sampling time scale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on nonradical reactions. The short footprinting time scale of FPOP offers two major advantages for protein structure elucidation: (1) it allows the protein to be interrogated in its native or near-native state with minimum structural perturbation; (2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addition, the covalent and irreversible oxidation by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS analysis, permitting high spatial resolution with residue-specific information. Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophysical tool for interrogating protein structure. In this Account, we summarize the principles and experimental design of FPOP that enable its fast labeling and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid β self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab, and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Additionally, the utility of FPOP-based oxidative footprinting should grow with our continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical, and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity toward various types of amino acid residues, providing complementary insights into protein higher order structures for soluble proteins and ultimately for membrane-bound proteins.

Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation.

The enzyme imidazole glycerol phosphate synthase (IGPS) is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) stimulates millisecond (ms) timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C) reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent.

Probing Conformational Motions Underlying Anesthetic Drug Actions in a Ligand-Gated Ion Channel

General anesthetics and ethanol exert their functional effects on pentameric ligand-gated ion channels (pLGICs) by binding to distinct allosteric sites that are far from the agonist binding sites. While recent high-resolution structures of prokaryotic and eukaryotic pLGICs, solved in the presence of these drugs have illuminated their binding sites, the structural mechanisms underlying how these drugs enhance or inhibit pLGIC function are largely unknown. Recently, a single residue variant in the bacterial pLGIC, GLIC, was identified that is potentiated by ethanol (PNAS 108:12149-12154, 2011). Taking advantage of this GLIC ethanol-sensitive mutant, we are using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy to study motions underlying ethanol potentiation of GLIC currents. Previous studies have shown that agonist-mediated channel opening is associated with an inward tilting of the extracellular binding domain (ECD). Here, we examined if, and how, the ECD moves in response to ethanol binding in the transmembrane channel domain. Individual cysteine residues were introduced in the ECD of an ethanol-sensitive GLIC construct (F14’A) at the top of β-strands (β1, 2, 5, 6, 8), labeled with the spin probe MTSL and ethanol-induced motions were measured using double electron-electron resonance (DEER) spectroscopy and compared to agonist (proton)-induced gating motions. DEER distance distributions for spin labels attached to N19C (β1), S93C (β5) and Q101C (β6) revealed that the attached probes moved closer together at pH 3.0, indicating that the inner β-sheet of GLIC ECD tilts inwards upon agonist activation. Interestingly, DEER distance distributions for N19 (β1) and S93 (β5) showed no significant changes in the presence of ethanol, indicating that structural mechanisms underlying ethanol allosteric drug modulation of pLGIC function are different than agonist-mediated motions that lead to channel activation.

Hydration Behavior along the Folding Pathways of Trpzip4, Trpzip5 and Trpzip6.

The microscopic properties of water confined within different segments of Trpzip4 (TZ4), Trpzip5 (TZ5), and Trzpip6 (TZ6) have been compared for all the states characterized along their folding pathways. In particular, structural ordering, energetics, and dynamics of water have been examined as the peptide unfolds along the free energy landscape. It is observed that the structuring of tetrahedral network as well as translational and rotational motions of hydration waters confined within the strands and the turn regions are very different, revealing motional heterogeneity in small 16-residue trpzips. The polar and charged groups present at the peptide surface anchor to water molecules through hydrogen bonds and are responsible for differential hydration among various segments of the peptide, which is found to be correlated to their hydropathy values. The coherent collective dynamics of water is strongly coupled with conformational changes in the peptide since the trends observed in most of the computed quantities are in accordance with the folded and unfolded states classified along the folding pathway for all trpzips. The hydration behavior conform to the heterogeneity observed in the free energy landscape of stable TZ4 with four unfolded states as compared to more flexible TZ5 and TZ6 with two unfolded states each, in addition to the folded state. The hydration waters are observed to regulate the protein dynamics by continuous fluctuations in hydrogen bond network involving lateral side chains that inject conformational motions in the peptide to facilitate its unfolding. The implications of mutations on various aspects of hydration water dynamics including their impact on structural and dynamic organization of hydrogen bonds are also highlighted. Our studies affirm that topology of the free energy landscape is shaped by both spatial organization and dynamic transitions in hydration waters in addition to the conformational fluctuations in the peptide along the folding pathway.

Transport-Relevant Protein Conformational Dynamics and Water Dynamics on Multiple Time Scales in an Archetypal Proton Channel: Insights from Solid-State NMR.

The influenza M2 protein forms a tetrameric proton channel that conducts protons from the acidic endosome into the virion by shuttling protons between water and a transmembrane histidine. Previous NMR studies have shown that this histidine protonates and deprotonates on the microsecond time scale. However, M2's proton conduction rate is 10-1000 s-1, more than 2 orders of magnitude slower than the histidine-water proton-exchange rate. M2 is also known to be conformationally plastic. To address the disparity between the functional time scale and the time scales of protein conformational dynamics and water dynamics, we have now investigated a W41F mutant of the M2 transmembrane domain using solid-state NMR. 13C chemical shifts of the membrane-bound peptide indicate the presence of two distinct tetramer conformations, whose concentrations depend exclusively on pH and hence the charge-state distribution of the tetramers. High-temperature 2D correlation spectra indicate that these two conformations interconvert at a rate of ∼400 s-1 when the +2 and +3 charge states dominate, which gives the first experimental evidence of protein conformational motion on the transport time scale. Protein 13C-detected water 1H T2 relaxation measurements show that channel water relaxes an order of magnitude faster than bulk water and membrane-associated water, indicating that channel water undergoes nanosecond motion in a pH-independent fashion. These results connect motions on three time scales to explain M2's proton-conduction mechanism: picosecond-to-nanosecond motions of water molecules facilitate proton Grotthuss hopping, microsecond motions of the histidine side chain allow water-histidine proton transfer, while millisecond motions of the entire four-helix bundle constitute the rate-limiting step, dictating the number of protons released into the virion.

Prediction of Hot Spots at Myeloid Cell Leukemia-1-Inhibitor Interface Using Energy Estimation and Alanine Scanning Mutagenesis.

Myeloid cell leukemia 1 (Mcl1) is an antiapoptotic protein that plays central role in apoptosis regulation. Also, Mcl1 has the potency to resist apoptotic cues resulting in up-regulation and cancer cell protection. A molecular probe that has the potential to specifically target Mcl1 and thereby provoke its down-regulatory activity is very essential. The aim of the current study is to probe the internal conformational dynamics of protein motions and potential binding mechanism in response to a series of picomolar range Mcl1 inhibitors using explicit-solvent molecular dynamics (MD) simulations. Subsequently, domain cross-correlation and principal component analysis was performed on the snapshots obtained from the MD simulations. Our results showed significant differences in the internal conformational dynamics of Mcl1 with respect to binding affinity values of inhibitors. Further, the binding free energy estimation, using three different samples, was performed on the MD simulations and revealed that the predicted energies (ΔGmmgbsa) were in good correlation with the experimental values (ΔGexpt). Also, the energies obtained using all sampling models were efficiently ranked. Subsequently, the decomposition energy analysis highlighted the major energy-contributing residues at the Mcl1 binding pocket. Computational alanine scanning performed on high energy-contributing residues predicted the hot spot residues. The dihedral angle analysis using MD snapshots on the predicted hot spot residue exhibited consistency in side chain conformational motion that ultimately led to strong binding affinity values. The findings from the present study might provide valuable guidelines for the design of novel Mcl1 inhibitors that might significantly improve the specificity for new-generation chemotherapeutic agents.

Expanding Functionality of Atomic Force Microscopy with High-Speed Environmental Studies

ABSTRACTEnvironmental atomic force microscopy (AFM) study of brush macromolecules, polymer blends and bitumen was performed with regular and Quick Scan imaging. Condensation of different vapors on sample surface has induced swelling of hydrophilic domains that helps recognizing the components of heterogeneous compounds. High-resolution imaging of brush macromolecules was achieved in ethyl acetate vapor. Fast monitoring of aggregation/spreading of brush macromolecules revealed dynamics of conformational changes and molecular motion.

Dynamics of dehaloperoxidase-hemoglobin A derived from NMR relaxation spectroscopy and molecular dynamics simulation

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S2 = 0.87 ± 0.03 and S2 = 0.76 ± 0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τm is 9.49 ± 1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant Rex using the Lipari-Szabo model-free formalism. These include Lys3, Ile6, Leu13, Gln18, Arg32, Ser48, Met49, Thr56, Phe60, Arg69, Thr71 Cys73, Ala77, Asn81, Gly95, Arg109, Phe115, Leu127 and Met136, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O2, H2O and H2O2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.

Characterizing protein conformations by correlation analysis of coarse-grained contact matrices.

We have developed a method to capture the essential conformational dynamics of folded biopolymers using statistical analysis of coarse-grained segment-segment contacts. Previously, the residue-residue contact analysis of simulation trajectories was successfully applied to the detection of conformational switching motions in biomolecular complexes. However, the application to large protein systems (larger than 1000 amino acid residues) is challenging using the description of residue contacts. Also, the residue-based method cannot be used to compare proteins with different sequences. To expand the scope of the method, we have tested several coarse-graining schemes that group a collection of consecutive residues into a segment. The definition of these segments may be derived from structural and sequence information, while the interaction strength of the coarse-grained segment-segment contacts is a function of the residue-residue contacts. We then perform covariance calculations on these coarse-grained contact matrices. We monitored how well the principal components of the contact matrices is preserved using various rendering functions. The new method was demonstrated to assist the reduction of the degrees of freedom for describing the conformation space, and it potentially allows for the analysis of a system that is approximately tenfold larger compared with the corresponding residue contact-based method. This method can also render a family of similar proteins into the same conformational space, and thus can be used to compare the structures of proteins with different sequences.

Variational Principles in the Mechanics of Conformational Motions of Macromolecules in a Viscous Medium

The mechanics of the conformational motions of macromolecules due to rotations around the valence bonds in a viscous medium have been considered. The variational principles for the energy-dissipation rate during conformational motions in a viscous medium and the rate of the potential energy decrease of a macromolecule during conformational relaxation have been analyzed. The seeming contradiction between this principle and the principle of the minimum energy-dissipation rate is resolved. It is shown that the energy-dissipation rate must be optimal and minimal in order to simultaneously satisfy the conservation laws and fulfill the deterministic nature of classical trajectories. The generalization and analysis of the influence of thermal fluctuations and external forces on the variational principles for the conformational relaxation of macromolecules is carried out. A visual graphical geometric depiction has been developed using hyperspheres in the space of the velocities of chain nodes to describe conformational movements along many degrees of freedom in a viscous medium. The equipartition of the energy-dissipation rates (and the rates of potential energy decrease) among the conformational degrees of freedom is discussed.

Three-dimensional movements of the pectoral fin during yaw turns in the Pacific spiny dogfish, Squalus suckleyi.

ABSTRACTFish pectoral fins move in complex ways, acting as control surfaces to affect force balance during swimming and maneuvering. Though objectively less dynamic than their actinopterygian relatives, shark pectoral fins undergo complex conformational changes and movements during maneuvering. Asynchronous pectoral fin movement is documented during yaw turning in at least two shark species but the three-dimensional (3D) rotation of the fin about the body axes is unknown. We quantify the 3D actuation of the pectoral fin base relative to the body axes. We hypothesized that Pacific spiny dogfish rotate pectoral fins with three degrees of freedom relative to the body during volitional turning. The pectoral fin on the inside of the turn is consistently protracted, supinated and depressed. Additionally, turning angular velocity increased with increasing fin rotation. Estimated drag on the fin increased and the shark decelerated during turning. Based on these findings, we propose that Pacific spiny dogfish uses drag-based turning during volitional swimming. Post-mortem muscle stimulation revealed depression, protraction and supination of the pectoral fin through stimulation of the ventral and cranial pterygoideus muscles. These data confirm functional hypotheses about pectoral fin musculature and suggest that Pacific spiny dogfish actively rotate pectoral fins to facilitate drag-based turning.This article has an associated First Person interview with the first author of the paper.

Deciphering mechanism of conformationally controlled electron transfer in nitric oxide synthases.

Electron transfer is a fundamental process in life that is very often coupled to catalysis within redox enzymes through a stringent control of protein conformational movements. Mammalian nitric oxide synthase (NOS) proteins are redox flavo-hemoproteins consisting of multiple modular domains. The NOS enzyme is exquisitely regulated in vivo by its partner, the Ca2+ sensing protein calmodulin (CaM), to control production of nitric oxide (NO). The importance of functional domain motion in NOS regulation has been increasingly recognized. The significant size and flexibility of NOS is a tremendous challenge to the mechanistic studies. Herein recent applications of modern biophysical techniques to NOS problems have been critically analyzed. It is important to note that any current biophysical technique alone can only probe partial aspects of the conformational dynamics due to limitations in the technique itself and/or the sample preparations. It is necessary to combine the latest methods to comprehensively quantitate the key conformational aspects (conformational states and distribution, conformational change rates, and domain interacting interfaces) governing the electron transfer. This is to answer long-standing central questions about the NOS isoforms by defining how specific CaM-NOS interactions and regulatory elements underpin the distinct conformational behavior of the NOS isoform, which in turn determine unique electron transfer and NO synthesis properties. This review is not intended as comprehensive, but as a discussion of prospects that promise impact on important questions in the NOS enzymology field.

Structural Features and Domain Movements Controlling Substrate Binding and Cofactor Specificity in Class II HMG-CoA Reductase

The key mevalonate pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) uses the cofactor NAD(P)H to reduce HMG-CoA to mevalonate in the production of countless metabolites and natural products. Although inhibition of HMGR by statin drugs is well-understood, several mechanistic details of HMGR catalysis remain unresolved, and the structural basis for the wide range of cofactor specificity for either NADH or NADPH among HMGRs from different organisms is also unknown. Here, we present crystal structures of HMGR from Streptococcus pneumoniae (SpHMGR) alongside kinetic data of the enzyme's cofactor preferences. Our structure of SpHMGR bound with its kinetically preferred NADPH cofactor suggests how NADPH-specific binding and recognition are achieved. In addition, our structure of HMG-CoA-bound SpHMGR reveals large, previously unknown conformational domain movements that may control HMGR substrate binding and enable cofactor exchange without intermediate release during the catalytic cycle. Taken together, this work provides critical new insights into both the HMGR reaction mechanism and the structural basis of cofactor specificity.

Structural and Conformational Chemistry from Electrochemical Molecular Machines. Replicating Biological Functions. A Review.

Each constitutive chain of a conducting polymer electrode acts as a reversible multi-step electrochemical molecular motor: reversible reactions drive reversible conformational movements of the chain. The reaction-driven cooperative actuation of those molecular machines generates, or destroys, inside the film the free volume required to lodge/expel balancing counterions and solvent: reactions drive reversible film volume variations, which basic structural components are here identified and quantified from electrochemical responses. The content of the reactive dense gel (chemical molecular machines, ions and water) mimics that of the intracellular matrix in living functional cells. Reaction-driven properties (composition-dependent properties) and devices replicate biological functions and organs. An emerging technological world of soft, wet, reaction-driven, multifunctional and biomimetic devices and the concomitant zoomorphic or anthropomorphic robots is presented.

The cooperative actuation of multistep electrochemical molecular machines senses the working temperature: voltammetric study

Electrochemical reactions of conducting polymers drive conformational movements of the constitutive polymeric chains (molecular motors or molecular machines), generating or destroying the required free volume to exchange balancing counterions and solvent. The influence of the temperature on the reversible oxidation/reduction of a polypyrrole film in NaCl aqueous solutions was studied here by cyclic voltammetry. Under constant potential limits, constant scan rate and constant electrolyte concentration the extension of the reversible reaction, defined by the coulovoltammetric charge, increases for rising experimental temperatures. That means that the reaction charge is a function of, and senses, the working temperature. Based on the empirical chemical kinetics of the reactions a theoretical description was attained resulting in a sensing equation. The attained results could explain how muscles from cold-blooded animals work and are aware of the thermal working conditions.

Conformation Manipulation and Motion of a Double Paddle Molecule on an Au(111) Surface.

The molecular conformation of a bisbinaphthyldurene (BBD) molecule is manipulated using a low-temperature ultrahigh-vacuum scanning tunneling microscope (LT-UHV STM) on an Au(111) surface. BBD has two binaphthyl groups at both ends connected to a central durene leading to anti/syn/flat conformers. In solution, dynamic nuclear magnetic resonance indicated the fast interexchange between the anti and syn conformers as confirmed by density functional theory calculations. After deposition in a submonolayer on an Au(111) surface, only the syn conformers were observed forming small islands of self-assembled syn dimers. The syn dimers can be separated into syn monomers by STM molecular manipulations. A flat conformer can also be prepared by using a peculiar mechanical unfolding of a syn monomer by STM manipulations. The experimental STM dI/dV and theoretical elastic scattering quantum chemistry maps of the low-lying tunneling resonances confirmed the flat conformer BBD molecule STM production. The key BBD electronic states for a step-by-step STM inelastic excitation lateral motion on the Au(111) are presented requiring no mechanical interactions between the STM tip apex and the BBD. On the BBD molecular board, selected STM tip apex positions for this inelastic tunneling excitation enable the flat BBD to move controllably on Au(111) by a step of 0.29 nm per bias voltage ramp.