Entropic and enthalpic contributions to the chair-boat conformational transformation in dextran under single molecule stretching

The contribution of entropy and enthalpy to the chair-boat conformational changes (clicks) occurring during the force-extension of single molecules of an axially linked polysaccharide, dextran, was investigated. Experimental single molecule force-extension measurements were carried out by atomic force microscopy over the temperature range of 5-70 degrees C. This enabled the separation of the entropy and enthalpy components of the conformational change. The contribution of entropy to the Gibbs energy of the conformational transformation was found to be small (<12 J mol(-1) K(-1)), demonstrating that the click is largely (>89%) enthalpic in nature.     

Preorganization and reorganization as related factors in enzyme catalysis: the chorismate mutase case

In this paper a deeper insight into the chorismate-to prephenate-rearrangement, catalyzed by Bacillus subtilis chorismate mutase, is provided by means of a combination of statistical quantum mechanics/molecular mechanics simulation methods and hybrid potential energy surface exploration techniques. The main aim of this work is to present an estimation of the preorganization and reorganization terms of the enzyme catalytic rate enhancement. To analyze the first of these, we have studied different conformational equilibria of chorismate in aqueous solution and in the enzyme active site. Our conclusion is that chorismate mutase preferentially binds the reactive conformer of the substrate--that presenting a structure similar to the transition state of the reaction to be catalyzed--with shorter distances between the carbon atoms to be bonded and more diaxial character. With respect to the reorganization effect, an energy decomposition analysis of the potential energies of the reactive reactant and of the reaction transition state in aqueous solution and in the enzyme shows that the enzyme structure is better adapted to the transition structure. This means not only a more negative electrostatic interaction energy with the transition state but also a low enzyme deformation contribution to the energy barrier. Our calculations reveal that the structure of the enzyme is responsible for stabilizing the transition state structure of the reaction, with concomitant selection of the reactive form of the reactants. This is, the same enzymatic pattern that stabilizes the transition structure also promotes those reactant structures closer to the transition structure (i.e., the reactive reactants). In fact, both reorganization and preorganization effects have to be considered as the two faces of the same coin, having a common origin in the effect of the enzyme structure on the energy surface of the substrate.     

Analysis of chorismate mutase catalysis by QM/MM modelling of enzyme-catalysed and uncatalysed reactions

Chorismate mutase is at the centre of current controversy about fundamental features of biological catalysts. Some recent studies have proposed that catalysis in this enzyme does not involve transition state (TS) stabilization but instead is due largely to the formation of a reactive conformation of the substrate. To understand the origins of catalysis, it is necessary to compare equivalent reactions in different environments. The pericyclic conversion of chorismate to prephenate catalysed by chorismate mutase also occurs (much more slowly) in aqueous solution. In this study we analyse the origins of catalysis by comparison of multiple quantum mechanics/molecular mechanics (QM/MM) reaction pathways at a reliable, well tested level of theory (B3LYP/6-31G(d)/CHARMM27) for the reaction (i) in Bacillus subtilis chorismate mutase (BsCM) and (ii) in aqueous solvent. The average calculated reaction (potential energy) barriers are 11.3 kcal mol(-1) in the enzyme and 17.4 kcal mol(-1) in water, both of which are in good agreement with experiment. Comparison of the two sets of reaction pathways shows that the reaction follows a slightly different reaction pathway in the enzyme than in it does in solution, because of a destabilization, or strain, of the substrate in the enzyme. The substrate strain energy within the enzyme remains constant throughout the reaction. There is no unique reactive conformation of the substrate common to both environments, and the transition state structures are also different in the enzyme and in water. Analysis of the barrier heights in each environment shows a clear correlation between TS stabilization and the barrier height. The average differential TS stabilization is 7.3 kcal mol(-1) in the enzyme. This is significantly higher than the small amount of TS stabilization in water (on average only 1.0 kcal mol(-1) relative to the substrate). The TS is stabilized mainly by electrostatic interactions with active site residues in the enzyme, with Arg90, Arg7 and Glu78 generally the most important. Conformational effects (e.g. strain of the substrate in the enzyme) do not contribute significantly to the lower barrier observed in the enzyme. The results show that catalysis is mainly due to better TS stabilization by the enzyme.     

Transition structure selectivity in enzyme catalysis: a QM/MM study of chorismate mutase

Two different transition structures (TSs) have been located and characterized for the chorismate conversion to prephenate in Bacillus subtilis chorismate mutase by means of hybrid quantum-mechanical/molecular-mechanical (QM/MM) calculations. GRACE software, combined with an AM1/CHARMM24/TIP3P potential, has been used involving full gradient relaxation of the position of ca. 3300 atoms. These TSs have been connected with their respective reactants and products by the intrinsic reaction coordinate (IRC) procedure carried out in the presence of the protein environment, thus obtaining for the first time a realistic enzymatic reaction path for this reaction. Similar QM/MM computational schemes have been applied to study the chemical reaction solvated by ca. 500 water molecules. Comparison of these results together with gas phase calculations has allowed understanding of the catalytic efficiency of the protein. The enzyme stabilizes one of the TSs (TS_OHout) by means of specific hydrogen bond interactions, while the other TS (TS_OHin) is the preferred one in vacuum and in water. The enzyme TS is effectively more polarized but less dissociative than the corresponding solvent and gas phase TSs. Electrostatic stabilization and an intramolecular charge-transfer process can explain this enzymatically induced change. Our theoretical results provide new information on an important enzymatic transformation and the key factors responsible for efficient selectivity are clarified. Received: 25 March 2000 / Accepted: 7 August 2000 / Published online: 23 November 2000     

lux-marked Pseudomonas aeruginosa lipopolysaccharide production in the presence of rhamnolipid

Rhamnolipid produced by Pseudomonas aeruginosa during the late logarithmic growth phase has been reported to be an effective biosurfactant. In the presence of rhamnolipid, P. aeruginosa had a higher specific lipopolysaccharide production, which was attributed to the increase of carbon to nitrogen ratios owing to the increase of the solubility of hydrophobic hydrocarbons, favoring the synthesis of lipopolysaccharides. P. aeruginosa also had a higher ratio of lipopolysaccharides in the growth medium to those on cell surfaces than in the absence of rhamnolipid because the presence of rhamnolipid stimulated the release of lipopolysaccharides from P. aeruginosa cell outer membranes. The release of lipopolysaccharides from cell surfaces made P. aeruginosa exhibit a more hydrophobic surface, enhancing the accumulation of hydrophobic hydrocarbons on P. aeruginosa cell surfaces and consequently resulting in a higher growth rate.     

Conformational effects in enzyme catalysis: QM/MM free energy calculation of the 'NAC' contribution in chorismate mutase

The controversial 'near attack conformation'(NAC) effect in the important model enzyme chorismate mutase is calculated to be 3.8-4.6 kcal mol(-1) by QM/MM free energy perturbation molecular dynamics methods, showing that the NAC effect by itself does not account for catalysis in this enzyme.     

Preferential affinity of the components of liquid mixtures at a rigid non-polar surface: enthalpic and entropic driving forces

The modulation of the properties of lipid membranes by polyhydroxylated cosolutes such as sugars is a phenomenon of considerable biological, technological and medicinal relevance. A few years ago, we proposed the sugar-like mechanism--binding driven by the release of water molecules--as an attempt to rationalize the preferential affinity of carbohydrate molecules compared to water molecules for the surface of lipid bilayers, which is presumably related to the bioprotective action of these compounds. The goal herein is to gain a better understanding of the driving force underlying this mechanism, in terms of specific interactions or effects, as well as in terms of the energy-entropy partitioning. This is done in the simplest possible context of an apolar rigid-wall model representing the membrane, and mixtures of closely related and possibly artificial species in solution, namely monomers or dimers of Lennard-Jones particles, water with physical or reduced charges, and hydroxymethyl groups. The results indicate that although the sugar-like mechanism seems phenomenologically reasonable, the main driving force underlying this mechanism is not the entropy gain upon releasing water molecules into the bulk, as originally suggested, but rather the hydrophobic effect. Note that the latter effect is a generic concept and may in principle involve both a solvent release and an interaction component, depending on the solute considered.     

Simultaneous determination of Pseudomonas aeruginosa elastase, human leukocyte elastase and cathepsin G activities by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.     

The Detection of Pseudomonas aeruginosa Using the Quartz Crystal Microbalance

Abstract

The development of a piezoelectric immunosensor for the detection of Pseudomonas aeruginosa in milk and dairy samples was undertaken here. This was achieved primarily by optimising the system using ELISA, investigating capture, competitive and displacement assays. Results from ELISA supplied information on detection limits and linear ranges obtained with each assay. A displacement assay was chosen to be transferred to the piezoelectric system and the reduction in mass on the surface of the crystal due to antigen displacement was measured by recording the frequency changes of the quartz crystal microbalance. The linear range obtained was from 2x10⁶ cell/ml to 1x10⁸ cell/ml and the limit of detection was 100,000 cells. The system was also tested for cross reactivity with a non-specific antigen, Pseudomonas fluorescens.     

Transition state stabilization and substrate strain in enzyme catalysis: ab initio QM/MM modelling of the chorismate mutase reaction

To investigate fundamental features of enzyme catalysis, there is a need for high-level calculations capable of modelling crucial, unstable species such as transition states as they are formed within enzymes. We have modelled an important model enzyme reaction, the Claisen rearrangement of chorismate to prephenate in chorismate mutase, by combined ab initio quantum mechanics/molecular mechanics (QM/MM) methods. The best estimates of the potential energy barrier in the enzyme are 7.4-11.0 kcal mol(-1)(MP2/6-31+G(d)//6-31G(d)/CHARMM22) and 12.7-16.1 kcal mol(-1)(B3LYP/6-311+G(2d,p)//6-31G(d)/CHARMM22), comparable to the experimental estimate of Delta H(++)= 12.7 +/- 0.4 kcal mol(-1). The results provide unequivocal evidence of transition state (TS) stabilization by the enzyme, with contributions from residues Arg90, Arg7, and Arg63. Glu78 stabilizes the prephenate product (relative to substrate), and can also stabilize the TS. Examination of the same pathway in solution (with a variety of continuum models), at the same ab initio levels, allows comparison of the catalyzed and uncatalyzed reactions. Calculated barriers in solution are 28.0 kcal mol(-1)(MP2/6-31+G(d)/PCM) and 24.6 kcal mol(-1)(B3LYP/6-311+G(2d,p)/PCM), comparable to the experimental finding of Delta G(++)= 25.4 kcal mol(-1) and consistent with the experimentally-deduced 10(6)-fold rate acceleration by the enzyme. The substrate is found to be significantly distorted in the enzyme, adopting a structure closer to the transition state, although the degree of compression is less than predicted by lower-level calculations. This apparent substrate strain, or compression, is potentially also catalytically relevant. Solution calculations, however, suggest that the catalytic contribution of this compression may be relatively small. Consideration of the same reaction pathway in solution and in the enzyme, involving reaction from a 'near-attack conformer' of the substrate, indicates that adoption of this conformation is not in itself a major contribution to catalysis. Transition state stabilization (by electrostatic interactions, including hydrogen bonds) is found to be central to catalysis by the enzyme. Several hydrogen bonds are observed to shorten at the TS. The active site is clearly complementary to the transition state for the reaction, stabilizing it more than the substrate, so reducing the barrier to reaction.     

Differential transition-state stabilization in enzyme catalysis: quantum chemical analysis of interactions in the chorismate mutase reaction and prediction of the optimal catalytic field

Chorismate mutase is a key model system in the development of theories of enzyme catalysis. To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.     

Electron tunneling in single crystals of Pseudomonas aeruginosa azurins.

Rates of reduction of Os(III), Ru(III), and Re(I) by Cu(I) in His83-modified Pseudomonas aeruginosa azurins (M-Cu distance approximately 17 A) have been measured in single crystals, where protein conformation and surface solvation are precisely defined by high-resolution X-ray structure determinations: 1.7(8) x 10(6) s(-1) (298 K), 1.8(8) x 10(6) s(-1) (140 K), [Ru(bpy)2(im)(3+)-]; 3.0(15) x 10(6) s(-1) (298 K), [Ru(tpy)(bpy)(3+)-]; 3.0(15) x 10(6) s(-1) (298 K), [Ru(tpy)(phen)(3+)-]; 9.0(50) x 10(2) s(-1) (298 K), [Os(bpy)2(im)(3+)-]; 4.4(20) x 10(6) s(-1) (298 K), [Re(CO)3(phen)(+)] (bpy = 2,2'-bipyridine; im = imidazole; tpy = 2,2':6',2' '-terpyridine; phen = 1,10-phenanthroline). The time constants for electron tunneling in crystals are roughly the same as those measured in solution, indicating very similar protein structures in the two states. High-resolution structures of the oxidized (1.5 A) and reduced (1.4 A) states of Ru(II)(tpy)(phen)(His83)Az establish that very small changes in copper coordination accompany reduction but reveal a shorter axial interaction between copper and the Gly45 peptide carbonyl oxygen [2.6 A for Cu(II)] than had been recognized previously. Although Ru(bpy)2(im)(His83)Az is less solvated in the crystal, the reorganization energy for Cu(I) --> Ru(III) electron transfer falls in the range (0.6-0.8 eV) determined experimentally for the reaction in solution. Our work suggests that outer-sphere protein reorganization is the dominant activation component required for electron tunneling.     

Biosynthesis of Gold Nanoparticles and Its Effect against Pseudomonas aeruginosa

Antimicrobial resistance has posed a serious health concern worldwide, which is mainly due to the excessive use of antibiotics. In this study, gold nanoparticles synthesized from the plant Tinospora cordifolia were used against multidrug-resistant Pseudomonas aeruginosa. The active components involved in the reduction and stabilization of gold nanoparticles were revealed by gas chromatography–mass spectrophotometry(GC-MS) of the stem extract of Tinospora cordifolia. Gold nanoparticles (TG-AuNPs) were effective against P. aeruginosa at different concentrations (50,100, and 150 µg/mL). TG-AuNPs effectively reduced the pyocyanin level by 63.1% in PAO1 and by 68.7% in clinical isolates at 150 µg/mL; similarly, swarming and swimming motilities decreased by 53.1% and 53.8% for PAO1 and 66.6% and 52.8% in clinical isolates, respectively. Biofilm production was also reduced, and at a maximum concentration of 150 µg/mL of TG-AuNPs a 59.09% reduction inPAO1 and 64.7% reduction in clinical isolates were observed. Lower concentrations of TG-AuNPs (100 and 50 µg/mL) also reduced the pyocyanin, biofilm, swarming, and swimming. Phenotypically, the downregulation of exopolysaccharide secretion from P. aeruginosa due to TG-AuNPs was observed on Congo red agar plates     

In vitro photodynamic eradication of Pseudomonas aeruginosa in planktonic and biofilm culture

Photodynamic disinfection (PDD) is a nonantibiotic approach to treating drug-resistant bacterial infections. Pseudomonas aeruginosa , an opportunistic pathogen, is problematic because of its propensity to develop antibiotic resistance and its ability to secrete a protective biofilm matrix. This study examined the ability of PDD to eradicate planktonic and biofilm cultures of P. aeruginosa in vitro . Planktonic P. aeruginosa cultures were briefly exposed to a methylene blue-based photosensitizer formulation and subjected to energy doses ranging from 1.7 to 20.6 J cm⁻² using a 670 nm nonthermal diode laser. Biofilms were grown for 24 and 48 h and exposed to photosensitizer for 30 s before illumination with 13.2 or 26.4 J of energy. A single exposure of planktonic P. aeruginosa to photosensitizer at >15.5 J cm⁻² resulted in 100% eradication (>7 log₁₀ reduction from control), an effect that could be decreased significantly in the presence of the singlet oxygen quenchers l -tryptophan and sodium azide. Decreasing the energy dose below this threshold by varying both power density and illumination duration resulted in a dose-dependent decrease in bacterial kill. In addition, 24 h biofilm viability was reduced by 99% with single exposure and 99.9% with double exposure, while 48 h biofilm viability was reduced by >99.999% with both single and double exposures. This study shows that PDD is effective in eradicating planktonic and biofilm cultures of P. aeruginosa, supporting the concept that translation into clinical practice for indications such as otitis externa and wound disinfection is a viable option.     

Microrheology of bacterial biofilms in vitro: Staphylococcus aureus and Pseudomonas aeruginosa

The rheology of bacterial biofilms at the micron scale is an important step to understanding the communal lifecycles of bacteria that adhere to solid surfaces, as it measures how they mutually adhere and desorb. Improvements in particle-tracking software and imaging hardware have allowed us to successfully employ particle-tracking microrheology to measuring single-species bacterial biofilms, based on Staphlococcus aureus and Pseudomonas aeruginosa. By tracking displacements of the cells at a range of timescales, we separate active and thermal contributions to the cell motion. The S. aureus biofilms in particular show power-law rheology, in common with other dense colloidal suspensions. By calculating the mean compliance of S. aureus biofilms, we observe them becoming less compliant during growth, and more compliant during starvation. The biofilms are rheologically inhomogeneous on the micron scale, as a result of the strength of initial adhesion to the flow cell surface, the arrangement of individual bacteria, and larger-scale structures such as flocs of P. aeruginosa. Our S. aureus biofilms became homogeneous as a function of height as they matured: the rheological environment experienced by a bacterium became independent of how far it lived from the flow cell surface. Particle-tracking microrheology provides a quantitative measure of the "strength" of a biofilm. It may therefore prove useful in identifying drug targets and characterizing the effect of specific molecular changes on the micron-scale rheology of biofilms.     

Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis

Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix. Formation of these sessile communities and their inherent resistance to anti-microbial agents are the source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major role in development of ocular related infectious diseases in human namely microbial keratitis. Different approaches have been used for preventing biofilm related infections in health care settings. Many of these methods have their own demerits that include chemical based complications; emergent antibiotic resistant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial keratitis based on its potential anti-biofilm activity.     

The role of quorum sensing in the pathogenicity of the cunning aggressor Pseudomonas aeruginosa

Recent decades have revealed that many bacterial species are capable of communicating with each other, and this observation has been largely responsible for a paradigm shift in microbiology. Whereas it was previously believed that bacteria lived as individual cells, it is now acknowledged that bacteria preferentially live in communities in the form of primitive organisms in which the behavior of individual cells is coordinated by cell–cell communication, known as quorum sensing (QS). Bacteria use QS for regulation of the processes involved in their interaction with each other, their environment, and, particularly, higher organisms We have focused on Pseudomonas aeruginosa, an opportunistic pathogen producing more than 30 QS-regulated virulence factors. P. aeruginosa causes several types of nosocomial infection, and lung infection in cystic fibrosis (CF) patients. We review the role of QS in the protective mechanisms of P. aeruginosa and show how disruption of the QS can be used as an approach to control this cunning aggressor.