Statistical Improvement of rGILCC 1 and rPOXA 1B Laccases Activity Assay Conditions Supported by Molecular Dynamics.

Laccases (E.C. 1.10.3.2) are glycoproteins widely distributed in nature. Their structural conformation includes three copper sites in their catalytic center, which are responsible for facilitating substrate oxidation, leading to the generation of H2O instead of H2O2. The measurement of laccase activity (UL-1) results may vary depending on the type of laccase, buffer, redox mediators, and substrates employed. The aim was to select the best conditions for rGILCC 1 and rPOXA 1B laccases activity assay. After sequential statistical assays, the molecular dynamics proved to support this process, and we aimed to accumulate valuable insights into the potential application of these enzymes for the degradation of novel substrates with negative environmental implications. Citrate buffer treatment T2 (CB T2) (pH 3.0 ± 0.2; λ420nm, 2 mM ABTS) had the most favorable results, with 7.315 ± 0.131 UL-1 for rGILCC 1 and 5291.665 ± 45.83 UL-1 for rPOXA 1B. The use of citrate buffer increased the enzyme affinity for ABTS since lower Km values occurred for both enzymes (1.49 × 10-2 mM for rGILCC 1 and 3.72 × 10-2 mM for rPOXA 1B) compared to those obtained in acetate buffer (5.36 × 10-2 mM for rGILCC 1 and 1.72 mM for rPOXA 1B). The molecular dynamics of GILCC 1-ABTS and POXA 1B-ABTS showed stable behavior, with root mean square deviation (RMSD) values not exceeding 2.0 Å. Enzyme activities (rGILCC 1 and rPOXA 1B) and 3D model-ABTS interactions (GILCC 1-ABTS and POXA 1B-ABTS) were under the strong influence of pH, wavelength, ions, and ABTS concentration, supported by computational studies identifying the stabilizing residues and interactions. Integration of the experimental and computational approaches yielded a comprehensive understanding of enzyme-substrate interactions, offering potential applications in environmental substrate treatments.

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study.

BACKGROUND: Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others. METHODS: A real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K. RESULTS: In the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t1/2 values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an Ed value of 41.40 KJ mol- 1, a low variation of KM and Vmax, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K. CONCLUSIONS: Laccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t1/2 of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.

Revisiting the Dielectric Constant Effect on the Nucleophile and Leaving Group of Prototypical Backside SN2 Reactions: A Reaction Force and Atomic Contribution Analysis.

The solvent effect on the nucleophile and leaving group atoms of the prototypical F- + CH3Cl → CH3F + Cl- backside bimolecular nucleophilic substitution reaction (SN2) is analyzed employing the reaction force and the atomic contributions methods on the intrinsic reaction coordinate (IRC). Solvent effects were accounted for using the polarizable continuum solvent model. Calculations were performed employing 11 dielectric constants, ε, ranging from 1.0 to 78.5, to cover a wide spectrum of solvents. The reaction force data reveal that the solvent mainly influences the region of the IRC preceding the energy barrier, where the structural rearrangement to reach the transition state occurs. A detailed analysis of the atomic role in the reaction as a function of ε reveals that the nucleophile and the carbon atom are the ones that contribute the most to the energy barrier. In addition, we investigated the effect of the choice of nucleophile and leaving group on the ΔE0 and ΔE‡ of Y- + CH3X → YCH3 + X- (X, Y = F, Cl, Br, I) in aqueous solution. Our analysis allowed us to find relationships between the atomic contributions to the activation energy and leaving group ability and nucleophilicity.

Dynamics of Energy Transfer in a Conjugated Dendrimer Driven by Ultrafast Localization of Excitations.

Solar energy conversion starts with the harvest of light, and its efficacy depends on the spatial transfer of the light energy to where it can be transduced into other forms of energy. Harnessing solar power as a clean energy source requires the continuous development of new synthetic materials that can harvest photon energy and transport it without significant losses. With chemically-controlled branched architectures, dendrimers are ideally suited for these initial steps, since they consist of arrays of chromophores with relative positioning and orientations to create energy gradients and to spatially focus excitation energies. The spatial localization of the energy delimits its efficacy and has been a point of intense research for synthetic light harvesters. We present the results of a combined theoretical experimental study elucidating ultrafast, unidirectional, electronic energy transfer on a complex molecule designed to spatially focus the initial excitation onto an energy sink. The study explores the complex interplay between atomic motions, excited-state populations, and localization/delocalization of excitations. Our findings show that the electronic energy-transfer mechanism involves the ultrafast collapse of the photoexcited wave function due to nonadiabatic electronic transitions. The localization of the wave function is driven by the efficient coupling to high-frequency vibrational modes leading to ultrafast excited-state dynamics and unidirectional efficient energy funneling. This work provides a long-awaited consistent experiment-theoretical description of excited-state dynamics in organic conjugated dendrimers with atomistic resolution, a phenomenon expected to universally appear in a variety of synthetic conjugated materials.

Crown complexation of protonated amino acids: influence on IRMPD spectra.

We report infrared multiple photon dissociation (IRMPD) spectra for a series of crown-adducted, protonated amino acids, generated by electrospray ionization. The tight chelation of 18-crown-6 on the protonated NH(3)(+) moiety results in a considerable red shift of the NH(3)(+) stretch modes, notably the antisymmetric NH(3)(+) stretch. This is rationalized by a distortion of the NH(3)(+) normal mode potential energy surface, as verified by quantum chemical calculations. On the other hand, the local oscillator modes, such as the carboxylic acid OH stretch, indole NH stretch, and phenol OH stretches, remain well-resolved and are subject to minor and predictable blue shifts of 5-15 cm(-1). Other chemically diagnostic modes, such as the guanidine NH stretch and alcohol OH stretches, also have discernible band positions. Crucially, some of these diagnostic band positions have little to no overlap with one another and can hence be readily distinguished. In addition, the complexes are often found to efficiently photodissociate by neutral loss of 18-crown-6, particularly for higher-basicity amino acids. This in principle opens the door on multiplexing the IRMPD experiment, where the IR spectra of multiple precursors are recorded simultaneously.

Electrostatic potential as a tool to understand interactions between malaria vaccine candidate peptides and MHC II molecules.

One of the most important problems in vaccine development consists in understanding receptor-ligand interactions between Class II Major Histocompatibility Complex molecules (MHC II) and antigenic peptides involved in inducing an appropriate immune response. In this study, we used X-ray crystallography structural data provided by the HLA-DRß1*0301-CLIP peptide interaction to compare native non-immunogenic and specifically-modified immunogenic peptides derived from the malarial SALSA protein, by analyzing molecular electrostatic potential surfaces on the most important regions of the peptide binding groove (Pockets 1, 4, 6 and 9). Important differences were found on the electrostatic potential induced by these peptides, particularly in MHC II conserved residues: Qα9, Sα53, Nα62, Nα69, Yß30, Yß60, Wß61, Qß70, Kß71 and Vß86, the same ones involved in establishing hydrogen bonds between Class II molecule-peptide and the recognition by T cell receptor, it correlating well with the change in their immunological properties. The results clearly suggest that modifications done on the electrostatic potential of these amino acids could favor the induction of different immune responses and therefore, their identification could allow modifying peptides a priori and in silico, so as to render them into immunogenic and protection-inducers and hence suitable components of a chemically-synthesized, multi-antigenic, minimal subunit based vaccine.

Back-and-Forth Energy Transfer during Electronic Relaxation in a Chlorin-Perylene Dyad.

Donor-acceptor dyads represent a practical approach to tuning the photophysical properties of linear conjugated polymers in materials chemistry. Depending on the absorption wavelength, the acceptor and donor roles can be interchanged, and as such, the directionality of the energy transfer can be controlled. Herein, nonadiabatic excited state molecular dynamics simulations have been performed in an arylethylene-linked perylene-chlorin dyad. After an initial photoexcitation at the Soret band of chlorin, we observe an ultrafast sequential electronic relaxation to the lowest excited state. This process is accomplished through an efficient round-trip chlorin-to-perylene-to-chlorin energy transfer. It is characterized by successive intermittent localized and delocalized vibronic dynamics. Nonradiative relaxation takes place mainly through energy transfer events with perylene acting as a "heat sink" through which the nonradiative relaxation is efficiently funneled, and the excess energy is dispersed in a larger space of vibrational degrees of freedom. Thus, our findings suggest the use of donor-acceptor dyads as a useful strategy when one needs to deactivate an electronic excitation.

The any particle molecular orbital/molecular mechanics approach.

A computational scheme is proposed to broaden the range of applications of multicomponent methodologies for the study of local properties of big molecular systems existing in the gas phase and in solvated environments. This scheme extends the any particle molecular orbital (APMO) approach in the quantum mechanics/molecular mechanics (QM/MM) framework. As a first assessment of the performance of the proposed approach, we estimate the proton affinities (PAs) of seventy amines in the gas phase and the proton binding energies (PBEs) in the gas phase and in an explicitly solvated environment of the sixty-one protons present in the chignolin protein. These calculations are performed with the QM/MM versions of the APMO second-order proton propagator (APMO-PP2) and the APMO extended Koopmans' theorem (APMO-KT) approaches. Calculated PAs and PBEs show significant reductions in the computational effort with a reduced loss in accuracy. These results suggest that the APMO/MM scheme might be used as a low-cost multi-component alternative for studies of local properties in big molecular systems. Graphical Abstract QMMM regions and CPU times for the APMO/MM approach.

Coherent exciton-vibrational dynamics and energy transfer in conjugated organics.

Coherence, signifying concurrent electron-vibrational dynamics in complex natural and man-made systems, is currently a subject of intense study. Understanding this phenomenon is important when designing carrier transport in optoelectronic materials. Here, excited state dynamics simulations reveal a ubiquitous pattern in the evolution of photoexcitations for a broad range of molecular systems. Symmetries of the wavefunctions define a specific form of the non-adiabatic coupling that drives quantum transitions between excited states, leading to a collective asymmetric vibrational excitation coupled to the electronic system. This promotes periodic oscillatory evolution of the wavefunctions, preserving specific phase and amplitude relations across the ensemble of trajectories. The simple model proposed here explains the appearance of coherent exciton-vibrational dynamics due to non-adiabatic transitions, which is universal across multiple molecular systems. The observed relationships between electronic wavefunctions and the resulting functionalities allows us to understand, and potentially manipulate, excited state dynamics and energy transfer in molecular materials.

QM/MM molecular dynamics study of the galactopyranose → galactofuranose reaction catalysed by Trypanosoma cruzi UDP-galactopyranose mutase.

The enzyme UDP-Galactopyranose Mutase (UGM) catalyses the conversion of galactopyranose into galactofuranose. It is known to be critical for the survival and proliferation of several pathogenic agents, both prokaryotic and eukaryotic. Among them is Trypanosoma cruzi, the parasite responsible for Chagas' disease. Since the enzyme is not present in mammals, it appears as a promising target for the design of drugs to treat this illness. A precise knowledge of the mechanism of the catalysed reaction would be crucial to assist in such design. In this article we present a detailed study of all the putative steps of the mechanism. The study is based on QM/MM free energy calculations along properly selected reaction coordinates, and on the analysis of the main structural changes and interactions taking place at every step. The results are discussed in connection with the experimental evidence and previous theoretical studies.

Structures of biomolecular ions in the gas phase probed by infrared light sources.

Infrared (IR) spectroscopy of biomolecular ions combines mass spectrometry's high sensitivity and ability to analyze complex mixtures with the enhanced structural information available from vibrational spectroscopy. IR spectroscopy is in principle well placed to distinguish isomers and allow chemical classification of unknown molecules. This review gives an outline of current instrumentation, spectroscopic approaches, and potential bottlenecks. We discuss the most promising applications in bioanalytical mass spectrometry in view of recent experimental results, as well as future applications based on bioinformatics.

Variations in the electrostatic landscape of class II human leukocyte antigen molecule induced by modifications in the myelin basic protein peptide: a theoretical approach.

The receptor-ligand interactions involved in the formation of the complex between Class II Major Histocompatibility Complex molecules and antigenic peptides, which are essential for establishing an adaptive immunological response, were analyzed in the Class II Human Leukocyte Antigen (HLA)--Myelin Basic Protein (MBP) peptide complex (HLA-DRbeta1*1501-MBP) using a multipolar molecular electrostatic potential approach. The Human Leukocyte Antigen--peptide complex system was divided into four pockets together with their respective peptide fragment and the corresponding occupying amino acid was replaced by each of the remaining 19 amino acids. Partial atomic charges were calculated by a quantum chemistry approach at the Hatree Fock/3-21*G level, to study the behavior of monopole, dipole and quadrupole electrostatic multipolar moments. Two types of electrostatic behavior were distinguished in the pockets' amino acids: "anchoring" located in Pocket 1 and 4, and "recognition" located in Pocket 4 and 7. According to variations in the electrostatic landscape, pockets were ordered as: Pocket 1>Pocket 9>>Pocket 4 approximately Pocket 7 which is in agreement with the binding ability reported for Class II Major Histocompatibility Complex pockets. In the same way, amino acids occupying the polymorphic positions beta13R, beta26F, beta28D, beta9W, beta74A, beta47F and beta57D were shown to be key for this Receptor-Ligand interaction. The results show that the multipolar molecular electrostatic potential approach is appropriate for characterizing receptor-ligand interactions in the MHC-antigenic peptide complex, which could have potential implications for synthetic vaccine design.

tRNA structure from a graph and quantum theoretical perspective.

One of the objectives of theoretical biochemistry is to find a suitable representation of molecules allowing us to encode what we know about their structures, interactions and reactivity. Particularly, tRNA structure is involved in some processes like aminoacylation and genetic code translation, and for this reason these molecules represent a biochemical object of the utmost importance requiring characterization. We propose here two fundamental aspects for characterizing and modeling them. The first takes into consideration the connectivity patterns, i.e. the set of linkages between atoms or molecular fragments (a key tool for this purpose is the use of graph theory), and the second one requires the knowledge of some properties related to the interactions taking place within the molecule, at least in an approximate way, and perhaps of its reactivity in certain means. We used quantum mechanics to achieve this goal; specifically, we have used partial charges as a manifestation of the reply to structural changes. These charges were appropriately modified to be used as weighted factors for elements constituting the molecular graph. This new graph-tRNA context allow us to detect some structure-function relationships.