Enzyme immobilization studied through molecular dynamic simulations.

In recent years, simulations have been used to great advantage to understand the structural and dynamic aspects of distinct enzyme immobilization strategies, as experimental techniques have limitations in establishing their impact at the molecular level. In this review, we discuss how molecular dynamic simulations have been employed to characterize the surface phenomenon in the enzyme immobilization procedure, in an attempt to decipher its impact on the enzyme features, such as activity and stability. In particular, computational studies on the immobilization of enzymes using i) nanoparticles, ii) self-assembled monolayers, iii) graphene and carbon nanotubes, and iv) other surfaces are covered. Importantly, this thorough literature survey reveals that, while simulations have been primarily performed to rationalize the molecular aspects of the immobilization event, their use to predict adequate protocols that can control its impact on the enzyme properties is, up to date, mostly missing.

Unveiling the conformational landscape of achiral all-cis tert-butyl ß-peptoids.

The synthesis and conformational study of N-substituted ß-alanines with tert-butyl side chains is described. The oligomers prepared by submonomer synthesis and block coupling methods are up to 15 residues long and are characterised by amide bonds in the cis-conformation. A conformational study comprising experimental solution NMR spectroscopy, X-ray crystallography and molecular modeling shows that despite their intrinsic higher conformational flexibility compared to their α-peptoid counterparts, this family of achiral oligomers adopt preferred secondary structures including a helical conformation close to that described with (1-naphthyl)ethyl side chains but also a novel ribbon-like conformation.

Probing Liquid-Ordered and Disordered Phases in Lipid Model Membranes: A Combined Theoretical and Spectroscopic Study of a Fluorescent Molecular Rotor.

An integrated theoretical/experimental strategy has been applied to the study of environmental effects on the spectroscopic parameters of 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecular rotor. The computational part starts from the development of an effective force field for the first excited electronic state of DPAP and proceeds through molecular dynamics simulations in solvents of different polarities toward the evaluation of Stokes shifts by quantum mechanics/molecular mechanics (QM/MM) approaches. The trends of the computed results closely parallel the available experimental results thus giving confidence to the interpretation of new experimental studies of the photophysics of DPAP in lipid bilayers. In this context, results show unambiguously that both flexible dihedral angles and global rotations are significantly retarded in a cholesterol/DPPC lipid matrix with respect to the DOPC matrix, thus confirming the sensitivity of DPAP to probe different environments and, therefore, its applicability as a probe for detecting different structures and levels of plasma membrane organization.

Enabling Racemization of Axially Chiral Subphthalocyanine-Tetracyanobutadiene-Aniline Enantiomers by Triplet State Photogeneration.

In recent years, several tetracyanobuta-1,3-diene (TCBD) conjugates have been prepared by linking the tetracyano unit to various electroactive moieties. These push-pull conjugates, besides showing interesting physicochemical properties, are axially chiral, a feature arising from the restricted rotation around the central bond of the butadiene. Yet, only in a few cases, separation and isolation of the enantiomers have been successfully achieved, owing to the configurational lability of the corresponding enantiopure species. Herein, we report the first example of photo- and electroactive TCBD-based derivatives showing unprecedented configurational stability and a peculiar light-triggered enantiomer conversion mechanism enabled by triple-state photogeneration. These systems represent a nice addition to the fast-increasing arsenal of artificial, light-controllable molecular switches.

Relating circular dichroism to atomic structure by means of MD simulations and computed CD spectra with α-peptoids as an example.

Classical molecular dynamics simulations have been combined with quantum calculations of CD spectra in order to fruitfully relate the experimental CD spectra, not only to the overall conformation of chiral α-peptoids, but also to their structure at the atomic scale, including the dihedral feature of the backbone (ψ,φ) and the orientation of the chiral side-chain (χ1). These simulations have been performed up to the hexamer Ac-(stbe)6-CO2tBu. We have shown that the number of states has a significant impact on the shape of the spectrum below 215 nm. The number of states computed is also critical to simulate the spectra of long oligomers. While 10 to 20 states are sufficient to simulate the CD spectra of short oligomers, 100 states or more are mandatory to converge the CD spectral shape for longer oligomers. The conformational sampling and the analysis of the intramolecular interactions responsible for the specific folding of the objects have been jointly explored by means of Replica Exchange MD and DFT calculations.

In silico investigation of the interaction between the voltage-gated potassium channel Kv4.3 and its auxiliary protein KChIP1.

The voltage-gated potassium channel Kv4.3 plays a vital role in shaping the timing, frequency, and backpropagation of electrical signals in the brain and heart by generating fast transient currents at subthreshold membrane potentials in repetitive firing neurons. To achieve its physiological function, Kv4.3 is assisted by auxiliary ß-subunits that become integral parts of the native A-type potassium channels, among which there are the Kv channel-interacting proteins (KChIPs). KChIPs are a family of cytosolic proteins that, when coexpressed with Kv4, lead to higher current density, modulation of channel inactivation and faster recovery from inactivation, while the loss of KChIP function may lead to severe pathological states. Recently, the structural basis of the KChIP1-Kv4.3 interaction was reported by using two similar X-ray crystallographic structures, which supported a crucial role for KChIP1 in enhancing the stability of the Kv4.3 tetrameric assembly, thus helping the trafficking of the channel to the plasma membrane. Here, we investigate through fully atomistic simulations the structure and stability of the human Kv4.3 tetramerization (T1) domain in complex with KChIP1 upon specific mutations located in the first and second interfaces of the complex, as compared to the wild-type (WT). Our results nicely complement the available structural and biophysical information collected so far on these complex variants. In particular, the degree of structural deviations and energetic instability, from small to substantial, observed in these variants with respect to the WT model seems to parallel well the level of channel dysfunction known from electrophysiology data. Our simulations provide an octameric structure of the WT KChIP1-Kv4.3 assembly very similar to the known crystal structures, and, at the same time, highlight the importance of a previously overlooked site of interaction between KChIP1 and the Kv4.3 T1 domain.

Temperature Dependence of the Structure and Dynamics of a Dye-Labeled Lipid in a Planar Phospholipid Bilayer: A Computational Study.

Fluorescent probes are widely employed to label lipids for the investigation of structural and dynamic properties of model and cell membranes through optical microscopy techniques. Although the effect of tagging a lipid with an organic dye is generally assumed to be negligible, optically modified lipids can nonetheless affect the local lipid structure and, in turn, the lipid lateral mobility. To better assess this potential issue, all-atom (MD) molecular dynamics simulations have been performed to study structural and dynamic effects in a model DOPC membrane in the presence of a standard Rhodamine B-labeled DOPE lipid (RHB) as a function of temperature, i.e., 293 K, 303 K, and 320 K. As the temperature is increased, we observe similar changes in the structural properties of both pure DOPC and RHB-DOPC lipid bilayers: an increase of the area per lipid, a reduction of the membrane thickness and a decrease of lipid order parameters. The partial density profile of the RHB headgroups and their orientation within the lipid bilayer confirm the amphiphilic nature of the RHB fluorescent moiety, which mainly partitions in the DOPC glycerol backbone region at each temperature. Moreover, at all temperatures, our results on lipid lateral diffusion support a non-neutral role of the dye with respect to the unlabeled lipid mobility, thus suggesting important implications for optical microscopy studies of lipid membranes.

Interplay between lipid lateral diffusion, dye concentration and membrane permeability unveiled by a combined spectroscopic and computational study of a model lipid bilayer.

Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility in both cellular and model bilayers has been extensively investigated in recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat of fluorescence techniques is the need to use dye-labeled variants of the lipid of interest, thus potentially perturbing the structural and dynamic properties of the native species. Generally, the effect of the dye/tracer molecule is implicitly assumed to be negligible. Nevertheless, in view of the widespread use of optically modified lipids for studying lipid bilayer dynamics, it is highly desirable to well assess this point. Here, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations have been combined together to uncover subtle structural and dynamic effects in DOPC planar membranes enriched with a standard Rhodamine-labeled lipid. Our findings support a non-neutral role of the dye-labeled lipids in diffusion experiments, quantitatively estimating a decrease in lipid mobility of up to 20% with respect to the unlabeled species. Moreover, results highlight the existing interplay between dye concentration, lipid lateral diffusion and membrane permeability, thus suggesting possible implications for future optical microscopy studies of biophysical processes occurring at the membrane level.

Negative Impact of Carbapenem Methylation on the Reactivity of ß-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses.

The Enterococcus faecium l,d-transpeptidase (Ldtfm) mediates resistance to most ß-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldtfm is rapidly acylated by ß-lactams belonging to the carbapenem class but not by penams or cephems. We previously reported quantum calculations and kinetic analyses for Ldtfm and showed that the inactivation profile is not determined by differences in drug binding (KD [equilibrium dissociation constant] values in the 50 to 80 mM range). In this study, we analyzed the reaction of a Cys sulfhydryl with various ß-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn (cCys-Asn) to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain, thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the ß-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for nonmethylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron-withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of ß-lactams. We concluded that the rate of Ldtfm acylation is largely determined by the ß-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.

Exploring the Conformation of Mixed Cis- Trans α,ß-Oligopeptoids: A Joint Experimental and Computational Study.

The synthesis and conformational preferences of a set of new synthetic foldamers that combine both the α,ß-peptoid backbone and side chains that alternately promote cis- and trans-amide bond geometries have been achieved and addressed jointly by experiment and molecular modeling. Four sequence patterns were thus designed and referred to as cis-ß- trans-α, cis-α- trans-ß, trans-ß- cis-α, and trans-α- cis-ß. α- and ß NtBu monomers were used to enforce cis-amide bond geometries and α- and ß NPh monomers to promote trans-amides. NOESY and molecular modeling reveal that the trans-α- cis-ß and cis-ß- trans-α tetramers show a similar pattern of intramolecular weak interactions. The same holds for the cis-α- trans-ß and trans-ß- cis-α tetramers, but the interactions are different in nature than those identified in the trans-α- cis-ß-based oligomers. Interestingly, the trans-α- cis-ß peptoid architecture allows establishment of a larger amount of structure-stabilizing intramolecular interactions.

The role of Tat peptide self-aggregation in membrane pore stabilization: insights from a computational study.

It is widely accepted that endocytosis mediates the uptake of cationic cell penetrating peptides (CPPs) at relatively low concentrations (i.e. nano- to micromolar), while direct transduction across the plasma membrane comes into play at higher concentrations (i.e. micro- to millimolar). This latter process appears to depend on peptide-driven cellular processes, which in turn may induce local perturbations of plasma-membrane composition and/or integrity, and to be favored by peptide aggregation, especially into dimers. Besides, in most studies CPPs are tethered to fluorescent dyes in order to track peptide transduction events under the microscope, although often overlooking the possible role played by the dyes in assisting translocation. In an effort to provide some insights into the transduction process, here we report on a molecular dynamics (MD) simulation study of a prototype of the CPP family, namely the Tat11 arginine-rich motif. To be specific, the translocation of Tat11 across a purposely-created membrane pore, either or not covalently-linked to the tetramethylrhodamine-5-maleimide (TAMRA) dye and in both its monomeric and dimeric form, is analyzed in some detail. Results from several unconstrained and steered MD simulations, as well as energy decomposition analysis, nicely support the latest experimental evidence and help to shed light on key factors enabling peptide transduction. In particular, our study highlights the much slower translocation kinetics of Tat11 dimer in comparison to the single peptide, and therefore its enhanced capability to stabilize membrane pores. Notably, it also shows how TAMRA has overall negligible kinetic and energetic effects on peptide transduction, yet it promotes this process indirectly by favoring peptide aggregation.

Norovirus RNA-dependent RNA polymerase: A computational study of metal-binding preferences.

Norovirus (NV) RNA-dependent RNA polymerase (RdRP) is essential for replicating the genome of the virus, which makes this enzyme a key target for the development of antiviral agents against NV gastroenteritis. In this work, a complex of NV RdRP bound to manganese ions and an RNA primer-template duplex was investigated using X-ray crystallography and hybrid quantum chemical/molecular mechanical simulations. Experimentally, the complex crystallized in a tetragonal crystal form. The nature of the primer/template duplex binding in the resulting structure indicates that the complex is a closed back-tracked state of the enzyme, in which the 3'-end of the primer occupies the position expected for the post-incorporated nucleotide before translocation. Computationally, it is found that the complex can accept a range of divalent metal cations without marked distortions in the active site structure. The highest binding energy is for copper, followed closely by manganese and iron, and then by zinc, nickel, and cobalt. Proteins 2017; 85:1435-1445. © 2017 Wiley Periodicals, Inc.

Catalytic Mechanism of Peptidoglycan Deacetylase: A Computational Study.

Bacterial peptidoglycan deacetylase enzymes are potentially important targets for the design of new drugs. In pathogenic bacteria, they modify cell-wall peptidoglycan by removing the acetyl group, which makes the bacteria more resistant to the host's immune response and other forms of attack, such as degradation by lysozyme. In this study, we have investigated the mechanism of reaction of acetyl removal from a model substrate, the N-acetylglucosamine/N-acetylmuramic acid dimer, by peptidogylcan deacetylase from Helicobacter pylori. For this, we employed a range of computational approaches, including molecular docking, Poisson-Boltzmann electrostatic pKa calculations, molecular dynamics simulations, and hybrid quantum chemical/molecular mechanical potential calculations, in conjunction with reaction-path-finding algorithms. The active site of this enzyme is in a region of highly negative electrostatic potential and contains a zinc dication with a bound water molecule. In the docked enzyme-substrate complex, our pKa calculations indicate that in the most stable protonation states of the active site the zinc-bound water molecule is in its hydroxide form and that the adjacent histidine residue, His247, is doubly protonated. In addition, there are one or two excess protons, with the neighboring aspartate residues, Asp12 and/or Asp199, being protonated. Overall, we find five classes of feasible reaction mechanisms, with the favored mechanism depending heavily on the protonation state of the active site. In the major one-excess-proton form, the mechanism with the lowest barrier (84 kJ mol-1) involves an initial protonation of the substrate nitrogen, followed by nucleophilic attack of the zinc-bound hydroxide and rupture of the substrate's carbon-nitrogen bond. However, in the minor two-excess-proton form, four mechanisms are almost equienergetic (83-86 kJ mol-1), comprising both those that start with nitrogen protonation and those in which nucleophilic attack by hydroxide occurs first.

Hybrid Potential Simulation of the Acylation of Enterococcus faecium l,d-Transpeptidase by Carbapenems.

The l,d-transpeptidases, Ldts, catalyze peptidoglycan cross-linking in ß-lactam-resistant mutant strains of several bacteria, including Enterococcus faecium and Mycobacterium tuberculosis. Although unrelated to the essential d,d-transpeptidases, which are inactivated by the ß-lactam antibiotics, they are nevertheless inhibited by the carbapenem antibiotics, making them potentially useful targets in the treatment of some important diseases. In this work, we have investigated the acylation mechanism of the Ldt from E. faecium by the carbapenem, ertapenem, using computational techniques. We have employed molecular dynamics simulations in conjunction with QC/MM hybrid potential calculations to map out possible reaction paths. We have focused on determining the following: (i) the protonation state of the nucleophilic cysteine of the enzyme when it attacks; (ii) whether nucleophilic attack and ß-lactam ring-opening are concerted or stepwise, the latter occurring via an oxyanion intermediate; and (iii) the identities of the proton acceptors at the beginning and end of the reaction. Overall, we note that there is considerable plasticity in the mechanisms, owing to the significant flexibility of the enzyme, but find that the preferred pathways are ones in which nucleophilic attack of cysteine thiolate is concerted with ß-lactam ring-opening.

Characterization of the divalent metal binding site of bacterial polysaccharide deacetylase using crystallography and quantum chemical calculations.

Peptidoglycan deacetlyase (HP0310, HpPgdA) from the gram-negative pathogen Helicobacter pylori, is the enzyme responsible for a peptidoglycan modification that counteracts the host immune response. In a recent study, we determined the crystallographic structure of the enzyme, which is a homo-tetramer (Shaik et al., PloS One 2011;6:e19207). The metal-binding site, which is essential for the enzyme's catalytic activity, is visible within the structure, but we were unable to identify the nature of the metal itself. In this study, we have obtained a higher-resolution crystal structure of the enzyme, which shows that the ion bound is, in fact, zinc. Analysis of the structure of the four sites, one per monomer, and quantum chemical calculations of models of the site in the presence of different divalent metal ions show an intrinsic preference for zinc, but also significant flexibility of the site so that binding of other ions can eventually occur.

Helical ambivalency induced by point mutations.

BACKGROUND: Mutation of amino acid sequences in a protein may have diverse effects on its structure and function. Point mutations of even a single amino acid residue in the helices of the non-redundant database may lead to sequentially identical peptides which adopt different secondary structures in different proteins. However, various physico-chemical factors which govern the formation of these ambivalent helices generated by point mutations of a sequence are not clearly known. RESULTS: Sequences generated by point mutations of helices are mapped on to their non-helical counterparts in the SCOP database. The results show that short helices are prone to transform into non-helical conformations upon point mutations. Mutation of amino acid residues by helix breakers preferentially yield non-helical conformations, while mutation with residues of intermediate helix propensity display least preferences for non-helical conformations. Differences in the solvent accessibility of the mutating/mutated residues are found to be a major criteria for these sequences to conform to non-helical conformations. Even with minimal differences in the amino acid distributions of the sequences flanking the helical and non-helical conformations, helix-flanking sequences are found be more solvent accessible. CONCLUSIONS: All types of mutations from helical to non-helical conformations are investigated. The primary factors attributing such changes in conformation can be: i) type/propensity of the mutating and mutant residues ii) solvent accessibility of the residue at the mutation site iii) context/environment dependence of the flanking sequences. The results from the present study may be used to design de novo proteins via point mutations.

Capturing molten globule state of α-lactalbumin through constant pH molecular dynamics simulations.

The recently developed methods of constant pH molecular dynamics directly captures the correlation between protonation and conformation to probe protein structure, function, and dynamics. In this work, we investigate the effect of pH on the conformational properties of the protein human α-lactalbumin. Constant pH simulations at both acidic and alkaline medium indicate the formation of the molten globule state, which is in accordance with the previous experimental observations (especially, in acidic medium). The size of the protein measured by its radius of gyration (RG) exhibits a marked increase in both acidic and alkaline medium, which matches with the corresponding experimentally observed value of RG found in the molten globule. The probability of native contacts is also considerably reduced at acidic and basic pH as compared to that of native structure crystallized at neutral pH. The mean fractal dimension D2 of the protein records a sharp increase in basic medium as compared to those in neutral and acidic solutions implying a significant pH induced conformational change. The mean square fluctuations of all residues of the entire protein are found to increase by several folds in both acidic and basic medium, which may be correlated with the normalized solvent accessibility of the residues indicating role of solvent accessible surface area on protein internal dynamics. The helices comprising the α-domain of the protein are moderately preserved in the acidic and alkaline pH. However, the ß-sheet structures present in the ß-domain are completely disrupted in both acidic as well as basic pH.

Are ambivalent α-helices entropically driven?

This work is a first attempt to characterise the conformational preference of structurally ambivalent helices in terms of their backbone conformational entropy. Ambivalent sequences conform to two different secondary structures (helix-sheet or helix-random coil or sheet-random coil, etc.) in two different proteins. For variable ambivalent helices, the helical conformations are found to possess less conformational entropy as compared with their non-helical counterparts when the ϕ-ψ dihedral angle range of the entire peptide segment is used to calculate the backbone conformational entropy. The favourable number of native contacts is a primary stabilising factor for these helical conformations. However, an opposite trend is observed when the ϕ-ψ angles of the individual amino acids are used to calculate the backbone conformational entropy. The results show that these peptide segments are rather reluctant to form helices, but are driven to form helices due to the favourable number of native contacts and optimum range of ϕ-ψ angle of the segments. Both procedures are validated by applying on conserved helices in the non-redundant database and their corresponding counterparts in the Structural Classification of Proteins database. Although context is a major determinant in deciding conformations of ambivalent sequences, no significant difference in the conformational entropy of sequences flanking ambivalent helical sequences in helical and non-helical forms is observed in this study. The results may be useful in understanding the structural context and environmental factors which leads to the formation of ambivalent helices and designing de novo proteins.

Local order and mobility of water molecules around ambivalent helices.

Water on a protein surface plays a key role in determining the structure and dynamics of proteins. Compared to the properties of bulk water, many aspects of the structure and dynamics of the water surrounding the proteins are less understood. It is interesting therefore to explore how the properties of the water within the solvation shell around the peptide molecule depend on its specific secondary structure. In this work we investigate the orientational order and residence times of the water molecules to characterize the structure, energetics, and dynamics of the hydration shell water around ambivalent peptides. Ambivalent sequences are identical sequences which display multiple secondary structures in different proteins. Molecular dynamics simulations of representative proteins containing variable helix, variable nonhelix, and conserved helix are also used to explore the local structure and mobility of water molecules in their vicinity. The results, for the first time, depict a different water distribution pattern around the conserved and variable helices. The water molecules surrounding the helical segments in variable helices are found to possess a less locally ordered structure compared to those around their corresponding nonhelical counterparts and conserved helices. The long conserved helices exhibit extremely high local residence times compared to the helical conformations of the variable helices, whereas the residence times of the nonhelical conformations of the variable helices are comparable to those of the short conserved helices. This differential pattern of the structure and dynamics of water molecules in the vicinity of conserved/variable helices may lend valuable insights for understanding the role of solvent effects in determining sequence ambivalency and help in improving the accuracy of water models used in the simulations of proteins.

Structure of hydration water in proteins: a comparison of molecular dynamics simulations and database analysis.

Hydration layer water molecules play important structural and functional roles in proteins. Despite being a critical component in biomolecular systems, characterizing the properties of hydration water poses a challenge for both experiments and simulations. In this context we investigate the local structure of hydration water molecules as a function of the distance from the protein and water molecules respectively in 188 high resolution protein structures and compare it with those obtained from molecular dynamics simulations. Tetrahedral order parameter of water in proteins calculated from previous and present simulation studies show that the potential of bulk water overestimates the average tetrahedral order parameter compared to those calculated from crystal structures. Hydration waters are found to be more ordered at a distance between the first and second solvation shell from the protein surface. The values of the order parameter decrease sharply when the water molecules are located very near or far away from the protein surface. At small water-water distance, the values of order parameter of water are very low. The average order parameter records a maximum value at a distance equivalent to the first solvation layer with respect to the water-water radial distribution and asymptotically approaches a constant value at large distances. Results from present analysis will help to get a better insight into structure of hydration water around proteins. The analysis will also help to improve the accuracy of water models on the protein surface.