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Water play an important role in many structural and physicochemical properties of lyophilized proteins. Molecular dynamics simulations were employed to study the explicit water distributions on four structurally diversed proteins: insulin-like growth factor 1 (IGF1), immunoglobin G1 (IgG1), human serum albumin (HSA), and collagen. The MD simulations were combined with the literature data on water vapor sorption isotherms. To account for the heterogeneity of protein surface, the water molecules were classified into different groups according to the binding strengths. A mechanistic mathematical model was built to describe the type-II vapor sorption isotherms and successfully applied to all four model protein systems. Although commonly used Brunauer-Emmett-Teller (BET) theory has a good fitting to the experimental vapor sorption isotherms, the basic "monolayer" concept is not consistent with reality – covering too limited protein surface. Experimentally, several physicochemical properties did show a break point near the BET "monolayer" level. This study demonstrates that the water content threshold or BET "monolayer" is consistent with the onset of water cluster (n≥3) formation. Based on water distributions at different amino acid sidechains as well as the backbones, a simple formula was derived based on primary sequence and fractions of ordered secondary structures (i.e. alpha helix and beta sheet) to predict the BET "monolayer". We find that proteins with helical structural elements are more stable upon changes in water content compared to other protein architectures. [ABSTRACT FROM AUTHOR]

We recently reported the discovery of a novel protein stabilizing dipeptide, glycyl-D-asparagine, through a structure-based approach. As the starting hypothesis leading to the discovery, we postulated a stabilizing effect achieved by binding of the dipeptide to an aggregation prone region on the protein's surface. Here we present a detailed study of the interaction mechanism between the dipeptide and Interferon-alpha-2A (IFN) through the construction of a Markov state model from molecular dynamics trajectories. We identify multiple binding sites and compare these to aggregation prone regions. Additionally, we calculate the lifetime of the protein-excipient complex. If the excipient remained bound to IFN after administration, it could alter the protein's therapeutic efficacy. We establish that the lifetime of the complex between IFN and glycyl-D-asparagine is extremely short. Under these circumstances, stabilization by stoichiometric binding is consequently no impediment for a safe use of an excipient. [ABSTRACT FROM AUTHOR]

We present a new framework for the description of macromolecules subject to confining geometries. The two main ingredients are a new computational method and the definition of a new molecular size parameter. The computational method, hereafter referred to the confinement analysis from bulk structures (CABS), allows the computation of equilibrium partition coefficients as a function of confinement size solely based on a single sampling of the configuration space of a macromolecule in bulk. Superior in computational speed to previous computational methods, CABS is capable of handling slits, channels, and box confining geometries for all molecular architectures. The new molecular size parameter, hereafter referred to the steric exclusion radius Rs, is explicitly defined and computed for a number of rigid objects and flexible polymers. We suggest that Rs is the relevant molecular size parameter for characterization of spatial confinement effects on macromolecules. Results for the equilibrium partition coefficient in the weak confinement regime depend only on the ratio of Rs to the confinement size regardless of molecular details. [ABSTRACT FROM AUTHOR]

Molecular dynamics simulations of diglyceride monolayers at the air–water interface have been performed to study the dynamical behavior of these Langmuir layers at surface densities varying from 36.2 to 40.5 Å2/molecule. The monolayers are treated in full atomic detail, with the exception of methyl and methylene groups that are considered to be ‘‘anisotropic united atoms.’’ The presence of phase transitions are explored and correlated to the changes in the microstructure of the layer. Good agreement is found between the surface pressure–surface area (π-A) isotherms calculated in the simulations and determined in monolayer technique experiments. The diglyceride film shows a complex dynamical behavior during expansion. On expansion from a closely packed, highly ordered state, the film undergoes two phase transitions. The first transition, which occurs at 38.3 Å2/molecule, involves a ‘‘seesaw’’ mechanism of the sn-1 and sn-2 carboxyester groups. The latter forms the backbone of the diglyceride molecule at high surface pressure. This mechanism is governed by hydrophobic/hydrophilic forces. It is stabilized by a balance between inter- and intramolecular interactions. On expansion, the intermolecular interaction decreases, and at the first transition, the intramolecular interaction between the two chains are strong enough to cause an increase of the cross sectional area of the molecules. This swelling of the molecules is accompanied with an increase of intermolecular contributions. The second transition, which occurs at 39.8 Å2/molecule, is caused by tilting over nearest neighbors. [ABSTRACT FROM AUTHOR]

Monte Carlo simulations of mixtures of charged and multipolar hard spheres with equivalent diameters are presented. A solution with dipolar solvent at a state point considered earlier, with greatly different results, by Chan et al. [Mol. Phys. 66, 299 (1989)] and by Caillol et al. [Mol. Phys. 69, 199 (1990)] is re-examined. Structural, thermodynamic, and dielectric properties are determined and particle number dependence in these averages is examined. The rate of convergence is shown to be satisfactory for correlation functions, other than that of repulsive ion pairs, and for thermodynamic properties. These are in good agreement with hypernetted chain theory. The reliability of the Metropolis Monte Carlo technique for the evaluation of thermodynamic properties in conveniently small systems of this solution model is established. We also consider a solution in which the solvent has both dipolar and linear quadrupolar symmetry. Two smaller systems give structural and thermodynamic properties in good agreement with hypernetted chain theory. In a larger system ionic clustering occurs to produce very different solution structure. [ABSTRACT FROM AUTHOR]

Helmholtz free energies, internal energies, and pressures of mixtures of charged and dipolar hard spheres are obtained from Monte Carlo computer simulation. Ionic and solvent contributions to these properties of this electrolyte solution model are reported for a large number of states, with variations in total density, ionic concentration, and the ratio of ionic charge to solvent polarity. From this data we show that, in contrast to solution theories based on replacement of the solvent by a continuum with dielectric constant ε0, there is a correspondence in properties of states at fixed κ02=κ2ε0, where κ is the inverse Debye length. [ABSTRACT FROM AUTHOR]

Phospholipases are ubiquitous in nature and the target of significant research aiming at both their physiological roles and technical applications in e.g. the food industry. In the search for sensitive and selective phospholipase assays, we have focused on synthetic FRET (Förster resonance energy transfer) substrates. This has led to the development of a facile, easily scalable and low cost synthesis of fluorogenic phospholipids featuring the dansyl/dabcyl fluorophore/quencher-pair on the fatty acid ω-position and on the phosphatidylethanolamine head group, respectively. Hence, the two substrates lyso-(dansyl-FA)-GPE-dabcyl ( 6 ) and (dansyl-FA) 2 -GPE-dabcyl ( 7 ) were synthesized by a chemoenzymatic strategy, in which preparation of ( 6 ) further included a novel selective enzymatic esterification step. As proof of concept, activity of a handful of phospholipases, one from each of the PLA1, PLA2, PLC and PLD classes, were assayed using substrates ( 6 ) and ( 7 ), and the kinetic parameter k cat /K M was determined. The PLA1 (Lecitase Ultra™) was found to be highly active on both substrates, whereas the PLD (from white cabbage) had no activity, presumably due to steric effects associated with the dabcyl-functionalization of the head group. It was further substantiated that the substrates are specific towards phospholipase activity as the tested lipase (Lipolase™) showed close to zero activity. [ABSTRACT FROM AUTHOR]

We have performed a series of molecular dynamics simulations to study the interactions between the neurotransmitters (NTs) γ-aminobutyrate (GABA), glycine (GLY), acetylcholine (ACH) and glutamate (GLU) as well as the amidated/acetylated γ-aminobutyrate (GABA neu ) and the osmolyte molecule glycerol (GOL) with a dipalmitoylphosphatidylcholine (DPPC) bilayer. In agreement with previously published experimental data, we found the lowest membrane affinity for the charged molecules and a moderate affinity for zwitterionic and polar molecules. The affinity can be ranked as follows: ACH–GLU << GABA < GLY << GABA neu << GOL. The latter three penetrated the bilayer at most with the deepest location being close to the glycerol backbone of the phospholipids. Even at that position, these solutes were noticeably hydrated and carried ∼30–80% of the bulk water along. The mobility of hydration water at the solute is also affected by the penetration into the bilayer. Two time scales of exchanging water molecules could be determined. In the bulk phase, the hydration layer contains ∼20% slow exchanging water molecules which increases 2–3 times as the solutes entered the bilayer. Our results indicate that there is no intermediate exchange of slow moving water molecules from the solutes to the lipid atoms and vice versa . Instead, the exchange relies on the reservoir of unbounded (“free”) water molecules in the interfacial bilayer region. Results from the equilibrium simulations are in good agreement with the results from umbrella sampling simulations, which were conducted for the four naturally occurring NTs. Free energy profiles for ACH and GLU show a minimum of ∼2–3 kJ/mol close to the bilayer interface, while for GABA and GLY, a minimum of respectively ∼2 kJ/mol and ∼5 kJ/mol is observed when these NTs are located in the vicinity of the lipid glycerol backbone. The most important interaction of NTs with the bilayer is the charged amino group of NTs with the lipid phosphate group. [ABSTRACT FROM AUTHOR]

Abstract: Generation of thermodynamic data, here compressed liquid density and isothermal compressibility data, using molecular dynamics simulations is investigated. Five normal alkane systems are simulated at three different state points. We compare two main approaches to isothermal compressibilities: (1) fluctuation solution theory analysis of trajectories obtained from simulations to yield total correlation function integrals; and (2) the more commonly used fluctuation formula. The results show that the two approaches yield consistent values and consistent uncertainties. Also, the computations converge in approximately the same amount of time. This suggests that computation of total correlation function integrals is a route to isothermal compressibility, as accurate and fast as well-established benchmark techniques. A crucial step is the integration of the radial distribution function. To obtain sensible results, this integral must be evaluated quite accurately. A previously employed integration method fits a radial distribution function expression to data. A modification is suggested for improving the accuracy, so that more accurate (individual) total correlation function integrals are obtained. [Copyright &y& Elsevier]

Abstract: The ‘State Conditions Transferability’ category of IFPSC 2006 tests prediction of binary vapor–liquid isotherms for mixtures of ethanol and the refrigerant HFF-227ea (1,1,1,2,3,3,3-heptafluoropropane). We predict these isotherms using fluctuation solution theory (FST). The method is based on isobaric–isothermal molecular dynamics (NPT-MD) simulations, using force field parameters published in the literature and fitted CHARMM force field parameters. Systems studied previously [S. Christensen, G.H. Peters, F.Y. Hansen, J.P. O’Connell, J. Abildskov, Molecular Simulation 33 (2007) 449–457] comprise the nearly ideal benzene/methyl acetate system, and the less ideal benzene/ethanol system at ambient temperatures. Both are at low pressures and remote from the pure component critical points. For the IFPSC system, we have used the same method even though predictions are for conditions remote from those of the provided data, the pressures are elevated, and the temperatures are near the critical temperature of one of the components. We first describe the computational method and thermodynamic modeling for the entry submitted, which assumed the vapor was an ideal gas and no Poynting correction was included. Then we discuss the effects of using common modeling methods to estimate the effects of elevated pressures. [Copyright &y& Elsevier]

Abstract: Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl-substituents attached to the sn-1 position of the glycerol backbone. The synthesis of phospholipids 1 and 2 is based upon the construction of a key aldehyde intermediate 3 which locks the stereochemistry in the sn-2 position of the final phospholipids. The aldehyde functionality serves as the site for insertion of the allyl-substituents by a zinc mediated allylation. Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA2 activity measurements. Our results show that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis of phospholipid 2 is due to steric hindrance caused by the bulky side chain of the substrate allowing only limited access of water molecules to the active site. [Copyright &y& Elsevier]

Abstract: The structure and molecular packing density of a “mismatched” solute, 1-hexanol, in lipid membranes of dimyristoyl phosphatidylcholine (DMPC) was studied by molecular dynamics simulations. We found that the average location and orientation of the hexanol molecules matched earlier experimental data on comparable systems. The local density or molecular packing in DMPC–hexanol was elucidated through the average Voronoi volumes of all heavy (non-hydrogen) atoms. Analogous analysis was conducted on trajectories from simulations of pure 1-hexanol and pure (hydrated) DMPC bilayers. The results suggested a positive volume change, ΔV m, of 4 cm3 mol−1 hexanol partitioned at 310 K in good accordance with experimental values. Analysis of the apparent volumes of each component in the pure and mixed states further showed that ΔV m reflects a balance between a substantial increase in the packing density of the alcohol upon partitioning and an even stronger loosening in the packing of the lipid. Furthermore, analysis of Voronoi volumes along the membrane normal identifies a distinctive depth dependence of the changes in molecular packing. The outer (interfacial) part of the lipid acyl chains (up to C8) is stretched by about 4%. Concomitantly, the average lateral area per chain decreases and these two effects compensate so that the overall packing density in the outer region, where the hexanol molecules are located, remains practically constant. The core of the bilayer (C9–C13) is slightly thinned. The average lateral area per chain in this region expands, resulting in a looser packing density. The net effect in the core is a 2–3% decrease in density corresponding to a total volume increase of ∼14 cm3 mol−1 hexanol partitioned. [Copyright &y& Elsevier]

Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Å apo PTP1B structure has been obtained, showing four highly coordinated water molecules in the active-site pocket of the enzyme; hence, the active site is highly solvated in the apo state. Three of the water molecules are located at positions that approximately correspond to the positions of the phosphate O atoms of the natural substrate phosphotyrosine and form a similar network of hydrogen bonds. The active-site WPD-Ioop was found to be in the closed conformation, in contrast to previous observations of wild- type PTPs in the apo state, in which the WPD-Ioop is open. The closed conformation is stabilized by a network of hydrogen bonds. These results provide new insights into and understanding of the active site of PTP1B and form a novel basis for structure-based inhibitor design. [ABSTRACT FROM AUTHOR]

To study the flexibility of the substrate-binding site and in particular of Gln262, we have performed adiabatic conformational search and molecular dynamics simulations on the crystal structure of the catalytic domain of wild-type protein-tyrosine phosphatase (PTP) 1 B, a mutant PTP1BR47v,D48N,M258c,G259Q, and a model of the catalytically active form of PTPα. For each molecule two cases were modeled: the Michaelis-Menten complex with the substrate analogue p-nitrophenyl phosphate (p-PNPP) bound to the active site and the cysteine-phosphor complex, each corresponding to the first and second step of the phosphate hydrolysis. Analyses of the trajectories revealed that in the cysteine-phosphor complex of PTP1B, G1n262 oscillates freely between the bound phosphate group and G1y259 frequently forming, as observed in the crystal structure, a hydrogen bond with the backbone oxygen of G1y259. In contrast, the movement of G1n262 is restricted in PTPα and the mutant due to interactions with G1n259 reducing the frequency of the oscillation of G1n262 and thereby delaying the positioning of this residue for the second step in the catalysis, as reflected experimentally by a reduction in kcat. Additionally, in the simulation with the Michaelis-Menten complexes, we found that a glutamine in position 259 induces steric hindrance by pushing the G1n262 side chain further toward the substrate and thereby negatively affecting Km as indicated by kinetic studies. Detailed analysis of the water structure around G1n262 and the active site Cys215 reveals that the probability of finding a water molecule correctly positioned for catalysis is much larger in PTP1B than in PTP1BR47V,D48N,M258c,G259Q and PTPα, in accordance with experiments. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPα, PTPβ, PTP∊, CD45, LAR, PTP1B and SHP-1), using pNPP as substrate. Most noticeable is the increase in the turnover number for PTPβ with increasing pH and the weak pH-dependence of the turnover number of CD45. The kinetic data for PTPα-D1 and PTPα-D1D2 suggest that D2 affects the catalysis of pNPP. PTP∊ and the closely homologous PTPα behave differently. The Km data were lower for PTP∊ than those for PTPα, while the inverse was observed for the catalytic efficiencies. [Copyright &y& Elsevier]

Binding properties of the native Thermomyces lanuginosa lipase (Tll), the inactive mutant of Tll (S146A; active Ser146 mutated to Ala), and the non-glycosylated mutant of Tll (N33Q) were determined using fluorescence spectroscopy. Tll, S146A mutant and N33Q mutant show significant different binding behavior to phosphatidylcholine (PC) and phosphatidylglycerol (PG) liposomes. Generally, weaker association of lipase molecules is observed to PC liposomes than to PG liposomes. Strong lipase–lipid interactions are observed for the S146A mutant, which is less pronounced for Tll and the N33Q variant. Addition of fatty acid to PG liposomes reduces significantly the binding affinity of the lipases. This effect is less pronounced in fatty acid/PC liposomes. Although the catalytic activity of the N33Q mutant is comparable to Tll, the non-glycosylated variant shows generally lower binding affinity to PC or PG matrix than Tll. Addition of the substrate analog benzene boronic acid (BBA) increases the binding affinity of the S146A and N33Q mutants, while only small changes are observed for Tll suggesting that the dynamics of the active site lid influences the binding affinity and that the flexibility of the loop region 33–48 might contribute to the activation of the lipase. [Copyright &y& Elsevier]

Lipases are efficient catalysts for lipolytic reactions and require a lipid interface for optimal activity. To study the effect of small charged lipid aggregates on the behavior of these enzymes, we have performed molecular dynamics simulations on five different systems. The simulations carried out in periodic boundary conditions and including explicit water molecules were performed for 2 ns. The lipids were either fatty acid molecules or amines to present negatively or positively charged species. The lipids were partially (i.e. mixture of charged and uncharged lipids) or fully charged. The lipids are randomly placed closely to the active site lid. Our results indicate that in all simulations different lipids do not affect the overall protein structure. However, conformational changes and internal motions in the protein are significantly influenced by the presence of the lipid molecules. In an aqueous environment, protein motions are mainly concentrated in three segments, which are Lys53-Asn63, Ser83-Asn86 and the C-terminus. The former region is structurally conserved in the lipase family and has been proposed to be involved in the activation of lipases. Interestingly, in the presence of the lipid molecules these fluctuations are suppressed, which is partly due to the interaction of the C-terminus with the lipid molecules. This may suggest that the C-terminus (which is not conserved in the lipase family) may have an important role in recognizing and binding to different lipid surfaces. In the presence of lipid aggregates, fluctuations are observed in the loops Val97-Lys106, Arg202-Gly214 and Trp223-Thr252. In particular, the flexibility of the loop Val97-Lys106 may have fundamental implications for the activation of lipases. This region has been suggested as a possible ‘entering gate’ for substrate molecules to the binding cleft. [Copyright &y& Elsevier]

In this note we present molecular dynamics (MD) simulation results of the melting transition of a hexane monolayer adsorbed on graphite. The present study was motivated by the recent simulation results of the same system by Hansen et al. [J. Chem. Phys. 98, 4128 (1993)]. Using a United Atom model for the representation of the inter- and intramolecular interactions, and the standard Steele expression for the monolayer-substrate potential, the latter authors obtained a melting temperature for the monolayer-substrate potential, the latter authors obtained a melting temperature for the monolayer which is at odds with experiment by 25%. In contrast, we show that when Steele’s method to model adsorbate–substrate interactions, based on Lorentz–Berthelot’s rule, is used, the transition temperature is in good agreement with experiment. The weaker surface potential also causes a slight increase in the width of the tilt distribution of the molecular axes and a decrease in the number of gauche defects. © 1995 American Institute of Physics. [ABSTRACT FROM AUTHOR]

We describe a method for extending radial distribution functions obtained from molecular simulations of pure and mixed molecular fluids to arbitrary distances. The method allows total correlation function integrals to be reliably calculated from simulations of relatively small systems. The long-distance behavior of radial distribution functions is determined by requiring that the corresponding direct correlation functions follow certain approximations at long distances. We have briefly described the method and tested its performance in previous communications [R. Wedberg, J. P. O'Connell, G. H. Peters, and J. Abildskov, Mol. Simul. 36, 1243 (2010); Fluid Phase Equilib. 302, 32 (2011)], but describe here its theoretical basis more thoroughly and derive long-distance approximations for the direct correlation functions. We describe the numerical implementation of the method in detail, and report numerical tests complementing previous results. Pure molecular fluids are here studied in the isothermal-isobaric ensemble with isothermal compressibilities evaluated from the total correlation function integrals and compared with values derived from volume fluctuations. For systems where the radial distribution function has structure beyond the sampling limit imposed by the system size, the integration is more reliable, and usually more accurate, than simple integral truncation. [ABSTRACT FROM AUTHOR]

The confinement analysis from bulk structure (CABS) approach [Y. Wang et al., J. Chem. Phys. 128, 124904 (2008)] is extended to determine the depletion profiles of dilute polymer solutions confined to a slit or near an inert wall. We show that the entire spatial density distributions of any reference point in the polymer chain (such as the center of mass, middle segment, and end segments) can be computed as a function of the confinement size solely based on a single sampling of the configuration space of a polymer chain in bulk. Through a simple analysis based on the CABS approach in the case of a single wall, we prove rigorously that (i) the depletion layer thickness δ is the same no matter which reference point is used to describe the depletion profile and (ii) the value of δ equals half the average span (the mean projection onto a line) of the macromolecule in free solution. Both results hold not only for ideal polymers, as has been noticed before, but also for polymers regardless of details in molecular architecture and configuration statistics. [ABSTRACT FROM AUTHOR]

Structural and dynamic properties of water confined between two parallel, extended, either hydrophobic or hydrophilic crystalline surfaces of n-alkane C36H74 or n-alcohol C35H71OH, are studied by molecular dynamics simulations. Electron density profiles, directly compared with corresponding experimental data from x-ray reflectivity measurements, reveal a uniform weak de-wetting characteristic for the extended hydrophobic surface, while the hydrophilic surface is weakly wetted. These microscopic data are consistent with macroscopic contact angle measurements. Specific water orientation is present at both surfaces. The ordering is characteristically different between the surfaces and of longer range at the hydrophilic surface. Furthermore, the dynamic properties of water are different at the two surfaces and different from the bulk behavior. In particular, at the hydrophobic surface, time-correlation functions reveal that water molecules have characteristic diffusive behavior and orientational ordering due to the lack of hydrogen bonding interactions with the surface. These observations suggest that the altered dynamical properties of water in contact with extended hydrophobic surfaces together with a partial drying of the surfaces are more indicative of the hydrophobic effect than structural ordering, which we suggest to be independent of surface topology. [ABSTRACT FROM AUTHOR]

Reducing the aggregation of proteins is of utmost interest to the pharmaceutical industry. Aggregated proteins are often less active and can cause severe immune reactions in the patient upon administration. At the same time the biopharmaceutical market is pushing for high concentration formulations and products that do not require refrigerated storage conditions. For a given protein, the only solution pH, ionic strength and concentration of a very limited number of excipients are the only parameters that can be varied to obtain a stable formulation. In this work, we present a structure-based approach to discover new molecules that successfully reduce the aggregation of proteins and apply the approach to the model protein Interferon-alpha-2a. [ABSTRACT FROM AUTHOR]

The sirtuins are NAD+‐dependent lysine deacylases, comprising seven isoforms (SIRT1–7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ‐N‐acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small‐angle X‐ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism‐based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA‐display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18. [ABSTRACT FROM AUTHOR]

The sirtuins are NAD+‐dependent lysine deacylases, comprising seven isoforms (SIRT1–7) in humans, which are involved in the regulation of a plethora of biological processes, including gene expression and metabolism. The sirtuins share a common hydrolytic mechanism but display preferences for different ϵ‐N‐acyllysine substrates. SIRT7 deacetylates targets in nuclei and nucleoli but remains one of the lesser studied of the seven isoforms, in part due to a lack of chemical tools to specifically probe SIRT7 activity. Here we expressed SIRT7 and, using small‐angle X‐ray scattering, reveal SIRT7 to be a monomeric enzyme with a low degree of globular flexibility in solution. We developed a fluorogenic assay for investigation of the substrate preferences of SIRT7 and to evaluate compounds that modulate its activity. We report several mechanism‐based SIRT7 inhibitors as well as de novo cyclic peptide inhibitors selected from mRNA‐display library screening that exhibit selectivity for SIRT7 over other sirtuin isoforms, stabilize SIRT7 in cells, and cause an increase in the acetylation of H3 K18. [ABSTRACT FROM AUTHOR]

Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration–response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration–response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underlying the drug-solubilizing properties of γ-cyclodextrin were explored using hydrocortisone as a model drug. The B S -type phase-solubility diagram of hydrocortisone with γ-cyclodextrin was thoroughly characterized by measuring the concentrations of hydrocortisone and γ-cyclodextrin in solution and the solid phase. The drug-solubilizer interaction was also studied by isothermal titration calorimetry from which a precise value of the 1:1 binding constant ( K 11 = 4.01 mM −1 at 20 °C) was obtained. The formation of water-soluble 1:1 complexes is responsible for the initial increase in hydrocortisone solubility while the precipitation of entities with a 3:2 ratio of γ-cyclodextrin:hydrocortisone is responsible for the plateau and the ensuing strong decrease in solubility once all solid hydrocortisone is used up. The complete phase-solubility diagram is well accounted for by a model employing the 1:1 binding constant and the solubility product of the precipitating 3:2 entity ( K 32 S = 5.51  mM 5 ) . For such systems, a small surplus of γ-cyclodextrin above the optimum concentration may result in a significant decrease in drug solubility, and the implications for drug formulations are briefly discussed. [ABSTRACT FROM AUTHOR]

We performed molecular dynamics simulations of Reteplase in the presence of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein–excipient formulations at room temperature, (ii) the ice-growth process, (iii)–(iv) the partially solvated and fully dried formulations, and (v) the reconstitution. Furthermore, coarse-grained (CG) simulations were employed to explore the protein-aggregation process in the presence of arginine. By using a coarse-grained representation, we could observe the collective behavior and interactions between protein molecules during the aggregation process. The CG simulations revealed that the presence of arginine prevented intermolecular interactions of the catalytic domain of Reteplase, thus reducing the aggregation propensity. This suggests that arginine played a stabilizing role by interacting with protein-specific regions. From the freeze-drying simulations, we could identify several protein-specific events: (i) collapse of the domain structure, (ii) recovery of the drying-induced damages during reconstitution, and (iii) stabilization of the local aggregation-prone region via direct interactions with excipients. Complementary to the simulations, we employed nanoDSF, size-exclusion chromatography, and CD spectroscopy to investigate the effect of the freeze-drying process on the protein structure and stability. [ABSTRACT FROM AUTHOR]

Fluorinated phospholipid analogues are investigated as potential substrates for phospholipase A2 (PLA2) using classical molecular dynamics simulations and quantum mechanics/density functional theory calculations. The fluorinated phospholipid analogues are α-fluoro (HF-ProAEL) and α,α-difluoro (F2-ProAEL) conjugates of (R)-1-O-hexadecyl-2-palmitoyl-sn-glycero-3-phoshocholineglycerol (ProAEL). Our results provide a theoretical assessment of the potential usefulness of these fluorinated lipids in the rational design of liposomal drug-delivery systems. The α-fluorine-substituted phospholipid analogues are found to be substrates for secretory PLA2, with sufficient accessibility of water to the active site to allow for enzymatic hydrolysis. Because of the inherently less stable nature of HF-ProAEL and F2-ProAEL when compared to that of ProAEL, the hydrolytic reaction is predicted to occur at a progressively faster rate; the more electronegative substituent at the α-position effectively lowers the energy barrier for hydrolysis. We conclude that the partially fluorinated phospholipid analogues facilitate rational design of liposomal vesicles of phospholipid mixtures with desirable physicochemical properties and that are still subjects for important and pharmaceutically proven drug-delivery mechanisms. [ABSTRACT FROM AUTHOR]

Here, we propose an optimised protocol for controlling the initial water activity (aw) in organic reaction mixtures with soluble lipase to ensure reproducible and consistent enzyme activity measurements. Pre‐equilibration above saturated salt solutions was used to set aw of the reaction media, where aw was tracked with a hygrometer. Consistent stirring of the suspension and volume correction to compensate for solvent evaporation were found to be critical. The protocol was tested on Candida antarctica lipase B (CALB) in soluble and immobilised forms in three organic solvents (acetonitrile, methyl tert‐butyl ether, and hexane) at four different water activities ranging from 0.12 to 0.97. Soluble and immobilised CALB had similar aw‐profiles and showed the highest enzyme activity in hexane. The optimised protocol expands the possibility to study enzyme kinetics from immobilised enzymes to soluble enzymes. [ABSTRACT FROM AUTHOR]

The article presents a study which explores the Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system. It mentions that oral protease inhibitor nirmatrelvir as of key importance for prevention of severe coronavirus disease 2019. It discusses that findings of the study have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.

A recently developed membrane-mimetic model was applied to study membrane interaction and binding of the two anchoring C2-like discoidin domains of human coagulation factor VIIIa (FVIIIa), the C1 and C2 domains. Both individual domains, FVIII C1 and FVIII C2, were observed to bind the phospholipid membrane by partial or full insertion of their extruding loops (the spikes). However, the two domains adopted different molecular orientations in their membrane-bound states; FVIII C2 roughly was positioned normal to the membrane plane, while FVIII C1 displayed a multitude of tilted orientations. The results indicate that FVIII C1 may be important in modulating the orientation of the FVIIIa molecule to optimize the interaction with FIXa, which is anchored to the membrane via its γ-carboxyglutamic acid-rich (Gla) domain. Additionally, a structural change was observed in FVIII C1 in the coiled main chain leading the first spike. A tight interaction with one lipid per domain, similar to what has been suggested for the homologous FVa C2, is characterized. Finally, we rationalize known FVIII antibody epitopes and the scarcity of documented hemophilic missense mutations related to improper membrane binding of FVIIIa, based on the prevalent nonspecificity of ionic interactions in the simulated membrane-bound states of FVIII C1 and FVIII C2. [ABSTRACT FROM AUTHOR]

Membranemechanical elastic properties regulate a variety of cellularprocesses involving local membrane deformation, such as ion channelfunction and vesicle fusion. In this work, we used molecular dynamicssimulations to estimate the local elastic properties of a membrane.For this, we calculated the energy needed to extract a DOPE lipidmolecule, modified with a linker chain, from a POPC bilayer membraneusing the umbrella sampling technique. Although the extraction energyentails several contributions related not only to elastic deformationbut also to solvation, careful analysis of the potential of mean force(PMF) allowed us to dissect the elastic contribution. With this information,we calculated an effective linear spring constant of 44 ± 4 kJ·nm–2·mol–1for the DOPC membrane,in agreement with experimental estimates. The membrane deformationprofile was determined independently during the stretching processin molecular detail, allowing us to fit this profile to a previouslyproposed continuum elastic model. Through this approach, we calculatedan effective membrane spring constant of 42 kJ·nm–2·mol–1, which is in good agreement with thePMF calculation. Furthermore, the solvation energy we derived fromthe data is shown to match the solvation energy estimated from criticalmicelle formation constants. This methodology can be used to determinehow changes in lipid composition or the presence of membrane modifierscan affect the elastic properties of a membrane at a local level. [ABSTRACT FROM AUTHOR]

Abstract: The possibility for obtaining derivative properties for mixtures from integrals of spatial total and direct correlation functions obtained from molecular dynamics simulations is explored. Theoretically well-supported methods are examined to extend simulation radial distribution functions to long range so the integrals can converge. A previously published method developed for pure atomic fluids is here extended to handle simulations of molecular mixtures using all-atom force fields. We first test the method on simulations of Lennard-Jones/Stockmayer mixtures and show that that the results are consistent with an excess Helmholtz energy model fitted to available simulations. In addition, simulations of water/methanol and water/t-butanol mixtures have been carried out. The method yields results for partial molar volumes, activity coefficient derivatives, and individual correlation function integrals in reasonable agreement with smoothed experimental data. The proposed method for obtaining correlation function integrals is shown to perform at least as well as or better than two previously published approaches. [Copyright &y& Elsevier]

[Display omitted] High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei , widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments. [ABSTRACT FROM AUTHOR]

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes. [ABSTRACT FROM AUTHOR]

Development of peptide therapeutics generally involves screening of excipients that inhibit peptide-peptide interactions, hence aggregation, and improve peptide stability. We used the therapeutic peptide plectasin to develop a fast screening method that combines microscale thermophoresis titration assays and molecular dynamics simulations to relatively rank the excipients with respect to binding affinity and to study key peptide-excipient interaction hotspots on a molecular level, respectively. Additionally, 1H-13C-HSQC NMR titration experiments were performed to validate the fast screening approach. The NMR results are in qualitative agreement with results from the fast screening method demonstrating that this approach can be reliably applied to other peptides and proteins as a fast screening method to relatively rank excipients and predict possible excipient binding sites. [ABSTRACT FROM AUTHOR]

Background: Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure–function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme's stability and kinetics. Results: Two expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384QAo). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs' value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30–60% higher for the single deficient variants compared to the WT, and about twofold larger for the triple deficient enzyme. Molecular dynamic simulation of the N-glycan mutants TrCel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. Conclusions: The kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates. [ABSTRACT FROM AUTHOR]

Fusion technology is widely used in protein-drug development to increase activity, stability, and bioavailability of protein therapeutics. Fusion proteins, like any other type of biopharmaceuticals, need to remain stable during production and storage. Due to the high complexity and additional intramolecular interactions, it is not possible to predict the behavior of fusion proteins based on the behavior the individual proteins. Therefore, understanding the stability of fusion proteins on the molecular level is crucial for the development of biopharmaceuticals. The current study on the albumin-neprilysin (HSA-NEP) fusion protein uses a combination of thermal and chemical unfolding with small angle X-ray scattering and molecular dynamics simulations to show a correlation between decreasing stability and increasing repulsive interactions, which is unusual for most biopharmaceuticals. It is also seen that HSA-NEP is not fully flexible: it is present in both compact and extended conformations. Additionally, the volume fraction of each conformation changes with pH. Finally, the presence of NaCl and arginine increases stability at pH 6.5, but decreases stability at pH 5.0. [ABSTRACT FROM AUTHOR]

Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2 precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ∼45% five-coordinate sites that selectively react with O2 relative to the remaining six-coordinate sites. However, this reaction produces an FeIII species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2 to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes. [ABSTRACT FROM AUTHOR]

An acoustically levitated droplet has been used to collect synchrotron SAXS data on human serum albumin protein solutions up to a protein concentration of 400 mg ml−1. A careful selection of experiments allows for fast data collection of a large amount of data, spanning a protein concentration/solvent concentration space with limited sample consumption (down to 3 µL per experiment) and few measurements. The data analysis shows data of high quality that are reproducible and comparable with data from standard flow‐through capillary‐based experiments. Furthermore, using this methodology, it is possible to achieve concentrations that would not be accessible by conventional cells. The protein concentration and ionic strength parameter space diagram may be covered easily and the amount of protein sample is significantly reduced (by a factor of 100 in this work). Used in routine measurements, the benefits in terms of protein cost and time spent are very significant. [ABSTRACT FROM AUTHOR]

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 A° resolution, revealing a single-domain, monomeric parallel β-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel β-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His- 271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes. [ABSTRACT FROM AUTHOR]

The development of a new protein drug typically starts with the design, expression and biophysical characterization of many different protein constructs. The initially high number of constructs is radically reduced to a few candidates that exhibit the desired biological and physicochemical properties. This process of protein expression and characterization to find the most promising molecules is both expensive and time-consuming. Consequently, many companies adopt and implement philosophies, e.g. platforms for protein expression and formulation, computational approaches, machine learning, to save resources and facilitate protein drug development. Inspired by this, we propose the use of interpretable artificial neuronal networks (ANNs) to predict biophysical properties of therapeutic monoclonal antibodies i.e. melting temperature T m , aggregation onset temperature T agg , interaction parameter k D as a function of pH and salt concentration from the amino acid composition. Our ANNs were trained with typical early-stage screening datasets achieving high prediction accuracy. By only using the amino acid composition, we could keep the ANNs simple which allows for high general applicability, robustness and interpretability. Finally, we propose a novel "knowledge transfer" approach, which can be readily applied due to the simple algorithm design, to understand how our ANNs come to their conclusions. [ABSTRACT FROM AUTHOR]

The lipidation of peptide drugs is one strategy to obtain extended half‐lives, enabling once‐daily or even less frequent injections for patients. The half‐life extension results from a combination of self‐association and association with human serum albumin (albumin). The self‐association and association with albumin of two insulin analogues, insulin detemir and insulin degludec, were investigated by small‐angle X‐ray scattering (SAXS) and dynamic light scattering (DLS) in phenolic buffers. Detemir shows concentration‐dependent self‐association, with an equilibrium between hexamer, dihexamer, trihexamer and larger species, while degludec appears as a dihexamer independent of concentration. The solution structure of the detemir trihexamer has a bent shape. The stoichiometry of the association with albumin was studied using DLS. For albumin–detemir the molar stoichiometry was determined to be 1:6 (albumin:detemir ratio) and for albumin–degludec it was between 1:6 and 1:12 (albumin:degludec ratio). Batch SAXS measurements of a 1:6 albumin:detemir concentration series revealed a concentration dependence of complex formation. The data allowed the modelling of a complex between albumin and a detemir hexamer and a complex consisting of two albumins binding to opposite ends of a detemir dihexamer. Measurements of size‐exclusion chromatography coupled to SAXS revealed a complex between a degludec dihexamer and albumin. Based on the results, equilibria for the albumin–detemir and albumin–degludec mixtures are proposed. Solution structures of complexes between human serum albumin and the two lipidated insulin analogues detemir and degludec are presented and models for the equilibria are suggested. [ABSTRACT FROM AUTHOR]

Despite a very large number of bacterial exopolysaccharides have been reported, detailed knowledge on their molecular structures and associative interactions with proteins is lacking. Small-angle X-ray scattering, dynamic light scattering and analytical ultracentrifugation (AUC) were used to characterize the interactions of six lactic acid bacterial heteroexopolysaccharides (HePS-1–HePS-6) with β-lactoglobulin (BLG). Compared to free HePSs, a large increase in the X-ray radius of gyration R G , maximum length L and hydrodynamic diameter d H of HePS-1–HePS-4 mixed with BLG revealed strong aggregation, the extent of which depended on the compact conformation and degree of branching of these HePSs. No significant effects were observed with HePS-5 and HePS-6. Turbidity and AUC analyses showed that both soluble and insoluble BLG–HePS complexes were formed. The findings provide new insights into the role of molecular structures in associative interactions between HePSs and BLG which has relevance for various industrial applications. [ABSTRACT FROM AUTHOR]

We describe the synthesis of tetrahydroisoquinolines and tetrahydroisoquinolinium salts together with their pharmacological properties at various nicotinic acetylcholine receptors. In general, the compounds were α4β2 nAChR antagonists, with the tetrahydroisoquinolinium salts being more potent than the parent tetrahydroisoquinoline derivatives. The most potent α4β2 antagonist, 6c, exhibited submicromolar binding Ki and functional IC50 values and high selectivity for this receptor over the α4β4 and α3β4 nAChRs. Whereas the (S)-6c enantiomer was essentially inactive at α4β2, (R)-6c was a slightly more potent α4β2 antagonist than the reference β2-nAChR antagonist DHβE. The observation that the α4β2 activity resided exclusively in the (R)-enantiomer was in full agreement with docking studies. Several of tetrahydroisoquinolinium salts also displayed agonist activity at the α7 nAChR. Preliminary in vivo evaluation revealed antidepressant-like effects of both (R)-5c and (R)-6c in the mouse forced swim test, supporting the therapeutic potential of α4β2 nAChR antagonists for this indication. [ABSTRACT FROM AUTHOR]

The effect of initial water activity of MTBE (methyl tert-butyl ether) medium on CALB (Candida antarctica lipase B) catalyzed esterification reaction is investigated using experimental methods and classical molecular dynamics (MD) simulations. The experimental kinetic studies show that the initial reaction rate of CALB-catalyzed esterification reaction between butyric acid and ethanol decreases with increasing initial water activity of the medium. The highest rate of esterification is observed at the lowest water activity studied. MD simulations were performed to gain a molecular insight on the effect of initial water activity on the rate of CALB-catalyzed reaction. Our results show that hydration has an insignificant effect on the structure and flexibility of CALB. Rather, it appears that water molecules bind to certain regions ("hot spots") on the CALB surface and form clusters. The size of the water clusters at these hot spot regions gradually increase and expand with increasing water activity. Consequently, the surface area of CALB covered by the water molecules also increases. Specifically, our results indicate that a particular water cluster located close to the active site partially cover the binding pocket of substrate at high water activity. As a consequence, the effective concentration of substrate at the catalytic site decreases. Therefore, the reaction rate slows down with increasing water activity, which correlates well with the observed decrease in the experimentally determined initial reaction rate. [ABSTRACT FROM AUTHOR]

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide. [ABSTRACT FROM AUTHOR]

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration RG, cross-sectional radius of gyration RXS, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. Φ and Ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ² of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins. [ABSTRACT FROM AUTHOR]