Thermus thermophilus HB27 laccase (Tth-Lac) is a thermostable enzyme that contains a β-hairpin (Ala292-Gln307) covering the substrate entrance. We analyzed the role of this β-hairpin in the enzymatic activity of Tth-Lac through three β-hairpin mutants: two variants without the β-hairpin (C1Tth-Lac and C2Tth-Lac) and one with a partially modified β-hairpin (P1Tth-Lac). Enzymatic activity was assayed with different substrates with and without copper. C1Tth-Lac showed a higher dependency on copper, increasing its activity by 1600-fold for syringaldazine (SGZ). All mutants presented a higher activity than Tth-Lac with phenolic substrates in the presence of copper. The position of the signal associated with CuT2 also changed, as shown in EPR spectra. Elucidation of the crystal structure of P1Tth-Lac mutant (PDB: 9CPM) showed that the partial deletion of the β-hairpin did not significantly affect the overall tertiary structure compared to the wild-type (PDB: 2xu9) nor the coordination of the four internally bound Cu atoms. Higher B-factors of the residues downstream of the deletion indicate increased flexibility (Q307, G308, P309, S310) that were otherwise more ordered in the Tth-Lac structure. Redox potential experiments on platinum electrodes have shown that all proteins have high redox potential, a finding that could have significant implications in the field of protein research.

The design of bioelectrochemical interfaces (BEI) is an interesting topic that recently demands attention. The synergy between biomolecules and chemical components is necessary to achieve high molecular selectivity and sensitivity for the development of biosensors, synthesis of different compounds, or catalytic processes. For most BEI, the charge transfer process occurs in environments with particular chemical conditions; modeling these environments is a challenging task and requires multidisciplinary efforts. These interfaces can be composed of biomolecules, such as proteins, DNA, or more complex systems like microorganisms. Oxidoreductases enzymes are good candidates, among others, due to their catalytic activities and structural characteristics. In BEI, enzymes are immobilized on conductive surfaces to improve charge transfer processes. Covalent immobilization is the most common method to prolong lifetime or modulate the detection process. However, it is necessary to implement new methodologies that allow the selection of the best candidates for a more efficient design. Homology modeling of oxidoreductases combined with Molecular Dynamics (MD) simulation methods are alternative and already routinely used tools to investigate the structure, dynamics, and thermodynamics of biological molecules. Our motivation is to show different techniques of molecular modeling (Homology Modeling, Gaussian accelerated molecular dynamics, directed adaptive molecular dynamics and electrostatic surface calculations), and using horseradish peroxidase as a model to understand the interactions between biomolecules and gold nanoclusters (as current collector). Additionally, we present our previous studies considering molecular simulations and we discuss recent advances in biomolecular simulations aimed at biosensor design.

The negative impact of infectious diseases like COVID-19 on public health and the global economy is evident. This pandemic represents a significant challenge for the scientific community to develop new practical analytical methods for accurately diagnosing emerging cases. Due to their selectivity and sensitivity, new methodologies based on antigen/antibody interactions to detect COVID-19 biomarkers are necessary. In this context, the theoretical, computational modeling reduces experimental efforts and saves resources for rational biosensor design. This study proposes using molecular dynamics to predict the interactions between the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein simplified model and a set of highly characterized antibodies. The binding free energy of the antigen/antibody complexes was calculated for the simplified models and compared against the complete SARS-CoV-2 ectodomain to validate the methodology. The structural data derived from our molecular dynamics and end-point free energy calculations showed a positive correlation between both approximations, with a 0.82 Pearson correlation coefficient; t = 3.661, df = 3, p-value = 0.03522, with a 95% confident interval. Furthermore, we identified the interfacial residues that could generate covalent bonds with a specific chemical surface without perturbing the binding dynamics to develop highly sensitive and specific diagnostic devices. Communicated by Ramaswamy H. Sarma. Graphical Abstract

The practical implementation of enzyme bioelectrodes is limited by the inherent lability of proteins, which require mild conditions to maintain both catalytic activity and long‐term stability. The lack of standardized reference parameters and the inherent complexity of biointerfaces also hinder direct comparison among electrochemical enzyme systems. Here, we present a reproducible platform based on the rational structural design of a laccase bioelectrode. Using its crystallographic structure, we performed an in silico evaluation of solvent accessibility and thermal flexibility to identify surface‐exposed lysine residues suitable for multipoint covalent immobilization on aryl‐carboxylic functionalized carbon paper under mild conditions. This strategy creates a simplified biointerface in which stability is integrated as an intrinsic property of the electrode‐enzyme system, eliminating the need for complex nanostructured materials. After 24 h of interfacial protein stabilization, the modified electrode exhibited improved electrochemical kinetics and retained ≈80% of its electroactivity after thermal, mechanical, chemical, and electrochemical stress, and ≈40% after 40 days of storage. The bioelectrode operated as a cathode in both a zinc‐based hybrid cell and a Geobacter sulfurreducens bioelectrochemical system, demonstrating the operational versatility of this approach. This work establishes a structurally guided strategy for the development of robust, reproducible, and comparable biointerfaces for bioelectrochemical applications.