LL-37 is a membrane-active antimicrobial peptide (AMP) that could disrupt the integrity of bacterial membranes due to its inherent cationic and amphipathic nature. Developing a shorter derivative of a long peptide such as LL-37 is of great interest, as it can reduce production costs and cytotoxicity. However, more detailed information about the residual interaction between LL-37 and the membrane is required for further optimization. Previously, molecular dynamics simulation using mixed all-atom and united-atom force fields showed that LL-37 could penetrate the bilayer membrane. This study aimed to perform all-atom molecular dynamics simulations, highlighting the residual interaction of LL-37 with the simplest model of the bacterial membrane, POPE:POPG (2:1), and compare its interaction with the POPC, which represents the eukaryotic membrane. The result showed leucine–leucine as the leading residues of LL-37 that first contact the membrane surface. Then, the cationic peptide of LL-37 started to penetrate the membrane by developing salt bridges between positively charged amino acids, Lys–Arg, and the exposed phosphate group of POPE:POPG, which is shielded in POPC. Residues 18 to 29 are suggested as the core region of LL-37, as they actively interact with the POPE:POPG membrane, not POPC. These results could provide a basis for modifying the amino acid sequence of LL-37 and developing a more efficient design for LL-37 derivatives.

The thermostability of enzymes plays a significant role in the starch hydrolysis process in the industry. The structural difference between thermostable Bacillus licheniformis alpha-amylase (BLA) and thermolabile Aspergillus niger alpha-amylase (ANA) is interesting to be explored. This work aimed to study the thermostability-determining factor of BLA as compared to a non-thermostable enzyme, ANA, using molecular dynamics (MD) simulation at a high temperature. A 100 ns of classical MD, which was followed by 200 ns accelerated MD was conducted to explore the conformational changes of the enzyme. It is revealed that the intramolecular interactions through salt bridge interactions and the presence of calcium ions dominates the stability effect of BLA, despite the absence of a disulfide bond in the structure. These results should be useful in designing a thermostable enzyme that can be used in industrial processes.

This study aims to isolate the RNA-dependent RNA polymerase (RdRp) gene of the dengue virus (DENV) isolates from Indonesia, determine the serotype of the local DENV strain through homology modeling, and express the recombinant RdRp protein in Escherichia coli BL21 (DE3), as the target for anti-dengue drug development. We utilize reverse transcription- polymerase chain reaction (RT-PCR) and nested PCR for serotyping the DENV isolates, obtained from Hasan Sadikin Hospital, Indonesia. Then, followed by the extraction and purification of the RdRp amplicon for gene sequencing. The resulting RdRp gene sequences are translated into amino acid sequences, which are then used for protein modeling and the construction of a recombinant plasmid for RdRp protein expression. As the result, the RdRp gene, which belongs to the non-structural protein 5 (NS5) of DENV, was successfully isolated and sequenced from DENV RNA samples. Based on serotyping results and homology modeling, the serotype of the isolate DENV was identified as serotype-3. The isolated NS5-RdRp gene from the local strain was then inserted into the pET28a(+) plasmid and transformed into E. coli BL21(DE3) as the host cell. Recombinant NS5-RdRp was predominantly found in the insoluble fraction with a molecular weight of 72 kDa. Future studies on the development of an anti-dengue drug model that confirms the inhibition of the binding domain in the recombinant RdRp protein structure will be required to provide more insight and expand the development of a universal anti-dengue strategy in Indonesia.

Developing therapeutics such as neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential to halt the Covid-19 infection. However, antibody production is expensive and relatively inaccessible to many low-income countries. Therefore, a more efficient and smaller antibody fragment, such as a single-chain variable fragment (scFv), derived from a known neutralizing antibody structure, is of interest due to the lower cost of recombinant protein production and the ability to tailor scFvs against circulating viruses. In this study, we used computational design to create an scFv based on the structure of a known neutralizing antibody, S230, for SARS-CoV-1. By analyzing the interaction of S230 with the RBD of both SARS-CoV-1 and SARS-CoV-2, five mutations were introduced to improve the binding of the scFv to the RBD of SARS-CoV-2. These mutations were Ser32Thr, Trp99Val, Asn57Val, Lys65Glu, and Tyr106Ile. Molecular dynamics simulations were used to evaluate the stability and affinity of the designed scFv. Our results showed that the designed scFv improved binding to the RBD of SARS-CoV-2 compared to the original S230, as indicated by principal component analysis, distance analysis, and MM/GBSA interaction energy. Furthermore, a positive result in a spot test lateral flow assay of the expressed scFv against the RBD indicated that the mutations did not alter the protein's structure. The designed scFv showed a negative result when tested against human serum albumin as a negative control, indicating reasonable specificity. We hope that this study will be useful in designing a specific and low-cost therapeutic agent, particularly during early outbreaks when information on neutralizing antibodies is limited.Communicated by Ramaswamy H. Sarma

Carotenoids are essential metabolites involved in photosynthesis, cellular protection, pigmentation, and antioxidant activities. Phytoene synthase (PSY/CrtB) utilizes C20 substrates in carotenoid biosynthesis, whereas its structural homolog, dehydrosqualene synthase (CrtM), preferentially accepts C15 substrates. Although previous studies have identified CrtM mutations that expand substrate scope, the molecular basis of substrate discrimination in PSY/CrtB remains poorly understood, largely because of the absence of experimentally determined three-dimensional structures. Here, we integrated comparative sequence analysis, homology modeling, and molecular dynamics (MD) simulations to investigate the structural basis of substrate discrimination in PSY/CrtB. Comparative sequence analysis suggested distinct overall conservation landscapes in PSY/CrtB and CrtM, with 20 highly conserved positions shared between them and clustered around the catalytic core. MD simulations suggest that PSY models exhibit minimal differentiation under cross-ligand conditions, consistent with its greater conformational plasticity. Surface property analysis suggested hydrophobic patches and an amphipathic helix (Helix-13) in PSY that were preferentially conserved in PSY homologs relative to CrtM homologs. Taken together, our analyses suggest that greater conformational plasticity may facilitate the accommodation of C20 substrates in PSY and that its conserved hydrophobic surface architecture may shape its surface physicochemistry. These findings suggest that differences in substrate accommodation between PSY/CrtB and CrtM may reflect coordinated variation in conformational dynamics, pocket hydrophobicity, and surface architecture, rather than substantial alterations to the conserved catalytic core.