The abundance of intrinsic disorder in the protein realm and its role in a variety of physiological and pathological cellular events have strengthened the interest of the scientific community in understanding the structural and dynamical properties of intrinsically disordered proteins (IDPs) and regions (IDRs). Attempts at rationalizing the general principles underlying both conformational properties and transitions of IDPs/IDRs must consider the abundance of charged residues (Asp, Glu, Lys, and Arg) that typifies these proteins, rendering them assimilable to polyampholytes or polyelectrolytes. Their conformation strongly depends on both the charge density and distribution along the sequence (i.e., charge decoration) as highlighted by recent experimental and theoretical studies that have introduced novel descriptors. Published experimental data are revisited herein in the frame of this formalism, in a new and possibly unitary perspective. The physicochemical properties most directly affected by charge density and distribution are compaction and solubility, which can be described in a relatively simplified way by tools of polymer physics. Dissecting factors controlling such properties could contribute to better understanding complex biological phenomena, such as fibrillation and phase separation. Furthermore, this knowledge is expected to have enormous practical implications for the design, synthesis, and exploitation of bio-derived materials and the control of natural biological processes.

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.

Liquid–liquid phase separation (LLPS) is responsible for the formation of so-called membrane-less organelles (MLOs) that are essential for the spatio-temporal organization of the cell. Intrinsically disordered proteins (IDPs) or regions (IDRs), either alone or in conjunction with nucleic acids, are involved in the formation of these intracellular condensates. Notably, viruses exploit LLPS at their own benefit to form viral replication compartments. Beyond giving rise to biomolecular condensates, viral proteins are also known to partition into cellular MLOs, thus raising the question as to whether these cellular phase-separating proteins are drivers of LLPS or behave as clients/regulators. Here, we focus on a set of eukaryotic proteins that are either sequestered in viral factories or colocalize with viral proteins within cellular MLOs, with the primary goal of gathering organized, predicted, and experimental information on these proteins, which constitute promising targets for innovative antiviral strategies. Using various computational approaches, we thoroughly investigated their disorder content and inherent propensity to undergo LLPS, along with their biological functions and interactivity networks. Results show that these proteins are on average, though to varying degrees, enriched in disorder, with their propensity for phase separation being correlated, as expected, with their disorder content. A trend, which awaits further validation, tends to emerge whereby the most disordered proteins serve as drivers, while more ordered cellular proteins tend instead to be clients of viral factories. In light of their high disorder content and their annotated LLPS behavior, most proteins in our data set are drivers or co-drivers of molecular condensation, foreshadowing a key role of these cellular proteins in the scaffolding of viral infection-related MLOs.

Sericin, a silk-derived protein recovered as a by-product of the textile industry, is a renewable biomacromolecule with considerable potential for sustainable material development. Chemical modification represents an effective strategy for its valorization. In this study, acetylation using acetyl chloride (AcCl) was selected as a model reaction to systematically investigate the reactivity of sericin in an N,N-dimethylacetamide/lithium chloride (DMA/LiCl) solvent system and to evaluate the influence of reaction parameters on both the extent of functionalization and protein secondary structure. A Design of Experiments (DoE) strategy, comprising an initial full factorial screening followed by Box–Behnken optimization, was employed to investigate the effects of AcCl equivalents, sericin concentration, and LiCl content. A quantitative FTIR workflow based on constrained Gaussian deconvolution was developed to derive functionalization index (FI) and β-sheet index (BI) from both peak areas and peak intensities, enabling the simultaneous evaluation of chemical modification and structural organization. The exploratory screening identified AcCl as the dominant factor governing sericin functionalization. During the optimization phase, the FI models described the general response trends, whereas the BI was successfully represented by robust quadratic models (R² = 0.977–0.978; adjusted R² = 0.936–0.938), revealing significant linear, interaction, and quadratic effects, with LiCl concentration and sericin concentration playing key roles in governing structural organization. The analytical workflow was verified by reproducibility assessment and independent validation experiments. Overall, this study proposes a quantitative DoE–FTIR framework for systematically investigating sericin functionalization and its associated structural evolution, providing support for future studies aimed at sericin industrial valorization.

BackgroundKRAS mutations are major oncogenic drivers, yet state-independent pan-KRAS inhibitors capable of targeting both active (GTP-bound) and inactive (GDP-bound) states remain extremely scarce.PurposeThe purpose of this work is to evaluate the non-covalent, state-independent pan-KRAS inhibitory potential of rosmarinic acid (RA) and botanical extracts from Rosmarinus officinalis and Salvia officinalis.Study designOur integrated multidisciplinary approach combines chemical characterization (qNMR and UPLC-ESI-HRMS/MS), biophysical binding assays (STD-NMR), computational analyses (molecular docking and molecular dynamics simulations), and in vitro functional assays on KRAS-dependent cancer cell lines, and Caco-2 cell monolayers as a non-proliferating epithelial model.MethodsMetabolites were profiled by NMR and UPLC-ESI-HRMS/MS and RA was quantified using qNMR. Direct interaction of RA and extracts with KRAS in GDP- and GMPPCP-bound states was characterized by STD-NMR, while molecular docking was used to investigate the predicted binding sites of RA. Antiproliferative activity and selectivity were assessed on KRAS-mutant cancer cells versus quiescent Caco-2 monolayers.ResultsqNMR provided accurate quantification of RA in both extracts. STD-NMR demonstrated direct, non-covalent, and state-independent binding of RA to KRAS in both inactive and active-like states, while docking analyses supported the proposed binding modes. Purified RA and botanical extracts inhibited the proliferation of KRAS-mutant cancer cells while showing limited cytotoxicity toward non-proliferating Caco-2 epithelial monolayers.ConclusionRosmarinic acid represents a promising non-covalent, state-independent pan-KRAS lead candidate, supporting further investigation of its potential for KRAS-targeted drug development.

Sericin, a silk-derived protein recovered as a by-product of the textile industry, is a re-newable biomacromolecule with considerable potential for sustainable material devel-opment. Chemical modification represents an effective strategy for its valorization. In this study, acetylation using acetyl chloride (AcCl) was selected as a model reaction to systematically investigate the reactivity of sericin in an N,N-dimethylacetamide/lithium chloride (DMA/LiCl) solvent system and to evaluate the in-fluence of reaction parameters on both the extent of functionalization and protein sec-ondary structure. A Design of Experiments (DoE) strategy, comprising an initial full factorial screening followed by Box–Behnken optimization, was employed to investi-gate the effects of AcCl equivalents, sericin concentration, and LiCl content. A quanti-tative FTIR workflow based on constrained Gaussian deconvolution was developed to derive functionalization (FI) and β-sheet (BI) indexes from both peak areas and peak in-tensities, enabling the simultaneous evaluation of chemical modification and structural organization. The exploratory screening identified AcCl as the dominant factor govern-ing sericin functionalization. During the optimization phase, the FI models described the general response trends, whereas the BI was successfully represented by robust quadratic models (R² = 0.977–0.978; adjusted R² = 0.936–0.938), revealing significant line-ar, interaction, and quadratic effects, with LiCl concentration and sericin concentration playing key roles in governing structural organization. The analytical workflow was verified by reproducibility assessment and independent validation experiments. Over-all, this study proposes a quantitative DoE–FTIR framework for systematically investi-gating sericin functionalization and its associated structural evolution, providing a support for future studies aimed at sericin industrial valorization.