Eduardo F. Franca

Characterization of Chitin and Chitosan Molecular Structure in Aqueous Solution.

Molecular dynamics simulations have been used to characterize the structure of single chitin and chitosan chains in aqueous solutions. Chitin chains, whether isolated or in the form of a β-chitin nanoparticle, adopt the 2-fold helix with ϕ and φ values similar to its crystalline state. In solution, the intramolecular hydrogen bond HO3(n)···O5(n+1) responsible for the 2-fold helical motif in these polysaccharides is stabilized by hydrogen bonds with water molecules in a well-defined orientation. On the other hand, chitosan can adopt five distinct helical motifs, and its conformational equilibrium is highly dependent on pH. The hydrogen bond pattern and solvation around the O3 atom of insoluble chitosan (basic pH) are nearly identical to these quantities in chitin. Our findings suggest that the solubility and conformation of these polysaccharides are related to the stability of the intrachain HO3(n)···O5(n+1) hydrogen bond, which is affected by the water exchange around the O3-HO3 hydroxyl group.

Chitosan molecular structure as a function of N‐acetylation

Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle-like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N-acetyl groups in the particles. The polysaccharide chains of highly N-deacetylated particles where the N-acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two-fold helix and five-fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two-fold helix with ϕ and ψ values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N-acetyl groups are unevenly distributed. Systems with 60% or higher N-acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N-acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3-HO3((n)) ···O5((n +) (1)) hydrogen bond, which in turn controls particle aggregation.

Designing an enzyme-based nanobiosensor using molecular modeling techniques

Nanobiosensors can be built via functionalization of atomic force microscopy (AFM) tips with biomolecules capable of interacting with the analyte on a substrate, and the detection being performed by measuring the force between the immobilized biomolecule and the analyte. The optimization of such sensors may require multiple experiments to determine suitable experimental conditions for the immobilization and detection. In this study we employ molecular modeling techniques to assist in the design of nanobiosensors to detect herbicides. As a proof of principle, the properties of acetyl co-enzyme A carboxylase (ACC) were obtained with molecular dynamics simulations, from which the dimeric form in an aqueous solution was found to be more suitable for immobilization owing to a smaller structural fluctuation than the monomeric form. Upon solving the nonlinear Poisson-Boltzmann equation using a finite-difference procedure, we found that the active sites of ACC exhibited a positive surface potential while the remainder of the ACC surface was negatively charged. Therefore, optimized biosensors should be prepared with electrostatic adsorption of ACC onto an AFM tip functionalized with positively charged groups, leaving the active sites exposed to the analyte. The preferential orientation for the herbicides diclofop and atrazine with the ACC active site was determined by molecular docking calculations which displayed an inhibition coefficient of 0.168 μM for diclofop, and 44.11 μM for atrazine. This binding selectivity for the herbicide family of diclofop was confirmed by semiempirical PM6 quantum chemical calculations which revealed that ACC interacts more strongly with the herbicide diclofop than with atrazine, showing binding energies of -119.04 and +8.40 kcal mol(-1), respectively. The initial measurements of the proposed nanobiosensor validated the theoretical calculations and displayed high selectivity for the family of the diclofop herbicides.

The Use of Functionalized AFM tips as Molecular Sensors in the Detection of Pesticides

Atomic force spectroscopy, a technique derived from Atomic Force Microscopy (AFM), allowed us to distinguish nonspecific and specific interactions between the acetolactate synthase enzyme (ALS) and anti-atrazine antibody biomolecules and the herbicides imazaquin, metsulfuron-methyl and atrazine. The presence of specific interactions increased the adhesion force (F

The Molecular Structure and Conformational Dynamics of Chitosan Polymers: An Integrated Perspective from Experiments and Computational Simulations

© 2012 Cunha et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Molecular Structure and Conformational Dynamics of Chitosan Polymers: An Integrated Perspective from Experiments and Computational Simulations

Nanobiosensor for Diclofop Detection Based on Chemically Modified AFM Probes

Highly sensitive and selective functional nanobiobreaksensors are being developed because they have significant applications in the sustenance and conservation of natural resources and can be used in projects to identify degraded and contaminated areas (of both soil and water) and as environmental quality indicators. In the present study, a nanobiosensor was developed based on using theoretical models (molecular docking and molecular dynamics simulations) based on biomimicry of the action mechanism of herbicides in plants coupled with atomic force microscopy (AFM) tools. The herbicide molecules were detected at very low concentrations using a unique sensor construction: the AFM probes and the substrate were chemically functionalized to favor covalent bonding and promote molecular flexibility, as well as to achieve reproducible and accurate results. Computational methods were used to determine the binding energies associated with the enzyme-herbicide interactions, which were compared with experimental results for adhesion forces. The theoretical results showed that the diclofop herbicide could be assembled and attached onto the mica substrate surface and the ACCase enzyme on the AFM probe without damaging the diclofop molecule. The experimental results showed that using a specific agrochemical target molecule was more efficient than using other nonspecific agrochemicals. On average, there was a 90% difference between the values of specific recognition (diclofop) and nonspecific recognition (imazaquin, metsulfuron, and glyphosate). This result validated the selectivity and specificity of the nanobiosensor. The first evidence of diclofop detection by the AFM probe sensors has been presented in this paper.

Modeling the coverage of an AFM tip by enzymes and its application in nanobiosensors

A stochastic simulation of adsorption processes was developed to simulate the coverage of an atomic force microscope (AFM) tip with enzymes represented as rigid polyhedrons. From geometric considerations of the enzyme structure and AFM tip, we could estimate the average number of active sites available to interact with substrate molecules in the bulk. The procedure was exploited to determine the interaction force between acetyl-CoA carboxylase enzyme (ACC enzyme) and its substrate diclofop, for which steered molecular dynamics (SMD) was used. The theoretical force of (1.6±0.5) nN per enzyme led to a total force in remarkable agreement with the experimentally measured force with AFM, thus demonstrating the usefulness of the procedure proposed here to assist in the interpretation of nanobiosensors experiments.

Molecular modeling of enzyme attachment on AFM probes.

The immobilization of enzymes on atomic force microscope tip (AFM tip) surface is a crucial step in the development of nanobiosensors to be used in detection process. In this work, an atomistic modeling of the attachment of the acetyl coenzyme A carboxylase (ACC enzyme) on a functionalized AFM tip surface is proposed. Using electrostatic considerations, suitable enzyme-surface orientations with the active sites of the ACC enzyme available for interactions with bulk molecules were found. A 50 ns molecular dynamics trajectory in aqueous solution was obtained and surface contact area, hydrogen bonding and protein stability were analyzed. The enzyme-surface model proposed here with minor adjustment can be applied to study antigen-antibody interactions as well as enzyme immobilization on silica for chromatography applications.

Computational study of polymorphic structures of α- and β- chitin and chitosan in aqueous solution.

Chitin is a natural biopolymer and the second most abundant after cellulose. This polysaccharide can be found in the biomass in different polymorphic forms. Chitosan is one of the most important derivatives obtained from the deacetylation of chitin. In this work, Molecular Dynamics simulations of chitin and chitosan nanoparticles enabled us to evaluate their different conformation and solubility properties. The Molecular Dynamics simulations show that the arrangement of the chains of chitin and chitosan significantly affects the structural behavior of these biopolymers in aqueous solution.

Unbinding pathway energy of glyphosate from the EPSPs enzyme binding site characterized by Steered Molecular Dynamics and Potential of Mean Force

The quantification of herbicides in the environment, like glyphosate, is extremely important to prevent contamination. Nanobiosensors stands out in the quantization process, because of the high selectivity, sensitivity and short response time of the method. In order to emulate the detection of glyphosate using a specific nanobiossensor through an Atomic Force Microscope (AFM), this work carried out Steered Molecular Dynamics simulations (SMD) in which the herbicide was unbinded from the active site of the enzyme 5- enolpyruvylshikimate 3 phosphate synthase (EPSPS) along three different directions.After the simulations, Potential of Mean Force calculations were carried, from a cumulant expansion of Jarzynskis equation to obtain the profile of free energy of interaction between the herbicide and the active site of the enzyme in the presence of shikimate-3 substrate phosphate (S3P). The set of values for external work, had a Gaussian distribution. The PMF values ranged according to the directions of the unbindong pahway of each simulation, displaying energy values of 10.7, 14.7 and 19.5KJmol-1. The results provide a theoretical support in order to assist the construction of a specific nanobiossensor to quantify the glyphosate herbicide.