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Alternative splicing (AS) enables cells to produce multiple protein isoforms from single genes, fine-tuning protein function across numerous cellular processes. However, despite its biological importance, researchers lack effective tools to compare the domain composition of AS-derived protein isoforms because such comparisons require both structural data and specialized methods. Recent advances in AI-driven protein structure prediction, particularly AlphaFold2, now make accurate structural determination of splicing isoforms accessible, enabling functional AS analysis at the protein structure level. Here, we present IsoformMapper, a web resource that analyzes AS through network community analysis of protein structures. This approach captures 3D physical interactions between protein regions often missed by traditional domain analysis, enabling structural comparisons of isoforms across any biological system. We illustrate our tool by analyzing validated human Bcl-X protein isoforms, revealing how AS creates distinct community structures with antagonistic functional roles. As a proof of concept, we apply our tool to investigate how GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling regulates plant de-etiolation through alternative splicing in Arabidopsis. In response to light, gun1 shows alterations in spliceosome component expression, suggesting that GUN1 contributes to AS regulation of genes essential for photosynthetic establishment. The gun1 mutant displays altered splice variant ratios for PNSL2, CHAOS, and SIG5. Our tool reveals that these isoforms form distinct protein community structures, demonstrating how AS impacts protein function and validating IsoformMapper's practical value.

Protein enzymes are highly efficient catalysts that exhibit adaptability and selectivity under diverse biological conditions. In some organisms, such as bacteria, structurally similar enzymes, for instance, shikimate kinase (SK) and adenylate kinase (AK), coexist and act on chemically related ligands. This raises the question of whether these enzymes can accommodate and potentially react with each other’s ligands. In this study, we investigate the stability of non-cognate ligand binding in SK and explore whether external electric fields (EFs) can modulate this interaction, leading to cross-reactivity in SK. Using molecular dynamics simulations, we assess the structural integrity of SK and the binding behavior of ATP and AMP under EF-off and EF-on cases. Our results show that EFs enhance protein structure stability, stabilize non-cognate ligands in the binding pocket, and reduce local energetic frustration near the R116 residue located in the binding site. In addition to this, dimensionality reduction analyses reveal that EFs induce more coherent protein motions and reduce the number of metastable states. Together, these findings suggest that external EFs can reshape enzyme–ligand interactions and may serve as a tool to modulate enzymatic specificity and functional promiscuity. Thus, we provide computational evidence that supports the concept of using an EF as a tunable parameter in enzyme engineering and synthetic biology. However, further experimental investigation would be valuable to assess the reliability of our computational predictions.

The intracellular environment displays complex dynamics influenced by factors such as molecular crowding and the low Reynolds number of the cytoplasm. Enzymes exhibiting active matter properties further heighten this complexity which can lead to memory effects. Molecular simulations often neglect these factors, treating the environment as a “thermal bath” using the Langevin equation (LE) with white noise. One way to consider these factors is by using colored noise instead within the generalized Langevin equation (GLE) framework, which allows for the incorporation of memory effects that have been observed in experimental data. We investigated the structural and dynamic differences in Shikimate kinase (SK) using LE and GLE simulations. Our results suggest that GLE simulations, which reveal significant changes, could be utilized for assessing conformational motions’ impact on catalytic reactions.

To produce chitosan is an interesting research. Chitosan is an important polysaccharide in terms of its various applications in industries and is produced from chitin, an abundant biopolymer in crustacean shell biomass wastes. Traditional processes for chitosan manufacture are commonly based on highly concentrated alkaline or acid solutions which are, however, severely eroding and harmful to the environment. In this study, we have described a ‘greener’ method using 1-ethyl-3-methylimidazolium acetate, [Emim][OAc] ionic liquid (IL), for decrystallization of shrimp crystalline chitin flakes followed by a microwave-mediated NaOH or tetrabutylammonium hydroxide, [TBA][OH], solution-based deacetylation for chitosan production. The decrease in crystallinity in IL pre-treated chitin was confirmed by XRD and SEM analysis which subsequently benefited chitosan production with up to 85% degree of deacetylation (%DDA) in shorter time periods (1-2 hours) and lower alkaline concentrations (20-40%). The %DDA in chitin/chitosan was estimated via FT-IR and NMR analysis. Notably, we could regenerate the ionic liquids: in case of [Emim][OAc] 97 wt.% and in case of [TBA][OH] 83 wt.% could be reused. Roles of ionic liquids in the process were discussed. Molecular dynamics (MD) simulations showed the roles of [TBA]+ cations in the molecular driving forces of [TBA][OH]-induced deacetylation mechanism. The strategy promises a sustainable and milder reaction approach to the existing highly corrosive alkaline- or acid-involved processes for chitosan production.

In its prefusion state, the SARS-CoV-2 spike protein (similarly to other class I viral fusion proteins) is metastable, which is considered to be an important feature for optimizing or regulating its functions. After the binding process of its S1 subunit (S1) with ACE2, the spike protein (S) undergoes a dramatic conformational change where S1 splits from the S2 subunit, which then penetrates the membrane of the host cell, promoting the fusion of the viral and cell membranes. This results in the infection of the host cell. In a previous work, we showed—using large-scale molecular dynamics simulations—that the application of external electric fields (EFs) induces drastic changes and damage in the receptor-binding domain (RBD) of the wild-type spike protein, as well of the Alpha, Beta, and Gamma variants, leaving a structure which cannot be recognized anymore by ACE2. In this work, we first extend the study to the Delta and Omicron variants and confirm the high sensitivity and extreme vulnerability of the RBD of the prefusion state of S to moderate EF (as weak as 104 V/m), but, more importantly, we also show that, in contrast, the S2 subunit of the postfusion state of the spike protein does not suffer structural damage even if electric field intensities four orders of magnitude higher are applied. These results provide a solid scientific basis to confirm the connection between the prefusion-state metastability of the SARS-CoV-2 spike protein and its susceptibility to be damaged by EF. After the virus docks to the ACE2 receptor, the stable and robust postfusion conformation develops, which exhibits a similar resistance to EF (damage threshold higher than 108 V/m) like most globular proteins.

Understanding the connection between local and global dynamics can provide valuable insights into enzymatic function and may contribute to the development of novel strategies for enzyme modulation. In this work, we investigated the dynamics at both the global and local (active site) levels of Shikimate Kinase (SK) through microsecond time-scale molecular dynamics (MD) simulations of the holoenzyme in the product state. Our focus was on the wild-type (WT) enzyme and two mutants (R116A and R116K) which are known for their reduced catalytic activity. Through exploring the dynamics of these variants, we gained insights into the role of residue R116 and its contribution to overall SK dynamics. We argue that the connection between local and global dynamics can be attributed to local frustration near the mutated residue which perturbs the global protein dynamics.

Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme’s conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme’s function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK’s conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme’s conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme’s active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

Shikimate kinase (SK) enzyme is a suitable target for antimicrobial drugs as it is present in pathogenic microorganisms and absent in mammals. A complete understanding of the functioning of this enzyme can unveil novel methods to inactivate it. To do this, a clear understanding of SK performance is needed. Previously, the chemical step of SK was studied in detail, but a study of longer-term scale simulations is still missing. In the present work, we performed molecular dynamics (MD) simulations in the µs time scale that allowed us to explore further regions of the SK energy landscape than previously. Simulations were conducted on the wild-type (WT) enzyme and the R116A and R116K mutants. We analyzed the dynamics of the enzymes through standard MD tools, and we found that the global motions in the mutants were perturbed. These motions can be linked to the observed undetectable binding affinity of the WT enzyme and the R116A and R116K mutants.

Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.