Flexible Region Research Articles

Proteolysis of β-Lactoglobulin Assisted by High Hydrostatic Pressure Treatment for Development of Polysaccharides-Peptides Based Coatings and Films

Peptides usually have many bioactive functions. The variety of peptide binding and the modularity of the components allow for their application to additional tissues and materials; hence broadening the range of possible coatings and films. β-lactoglobulin (b-LG) forms spherical microgels or can be used in the formation of coated particles, with the core formed by aggregated b-LG and the coat by polysaccharides. The enzymatic proteolysis of b-LG assisted by high hydrostatic pressure (HHP) treatment was studied. Pretreatment of HHP enhanced the hydrolysis degree (DH) of b-LG. The highest value of DH without pretreatment was 24.81% at 400 MPa, which increased to 27.53% at 200 MPa with pretreatment, suggesting a difference in the DH of b-LG caused by the processing strategy of HHP. Molecular simulation suggested that the flexible regions of b-LG, e.g., Leu140-Ala142 and Asp33-Arg40, might contribute to enzymatic proteolysis. The b-LG hydrolysate exhibited the highest capacity of scavenging free DPPH and OH radicals at 200 MPa. In addition, the 1–2 kDa and 500–1000 Da peptides fractions significantly increased from 10.53% and 9.78% (under 0.1 MPa) to 12.37% and 14.95% under 200 MPa, respectively. The higher yield of short peptides under HHP contributed to the antioxidant capacity of b-LG hydrolysates. Enzymatic hydrolysis also largely reduced the immunoreactivity of b-LG, which is of high importance in the practical application of b-LG in the field of coatings and films in regard to biocompatibility. Hydrolysis of b-LG assisted by high-pressure treatment showed promising potential in the preparation of bioactive peptides for further development of polysaccharide-peptide-based coatings and films.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.

An analytical nonlinear dynamic model for linear guide with carriage flexibility

Accurate estimation of vibration characteristics of linear guide is imperative for sophisticated structural performance of linear motion equipment. It has been illustrated that flexible model considering carriage deformation is a more precise description of realistic system. However, few dynamic model of carriage flexibility is found for vibration analysis when complex external load is applied. This paper proposes an analytical model to analyze the nonlinear vibration behavior of flexible linear guide system under multidirectional harmonic excitations. The outward deformation of flexible carriage skirt region is calculated by the classical deformation formula of vertical loaded cantilever beam. A lumped dynamic model accounting for segmented restoring force is proposed by coupling the vertical, lateral and rolling displacement and outward deformation. Dynamic responses of rigid and flexible models are compared, and the condition that neglects carriage flexibility is obtained. Numerical simulation exhibits abundant and fascinating nonlinear phenomena when the effects of external excitation, preload and carriage weight are discussed. Additionally, excitation experiments are performed to verify the proposed model. The developed theoretical model is promising for structure optimization of linear guide.

Biomechanical Effects of Proximal Polyetheretherketone Rod Extension on the Upper Instrumented and Adjacent Levels in a Human Long-Segment Construct: A Cadaveric Model.

ObjectiveThe high mechanical stress zone at the sudden transition from a rigid to flexible region is involved in proximal junctional kyphosis (PJK) physiopathology. We evaluated the biomechanical performance of polyetheretherketone (PEEK) rods used as a nontraditional long semirigid transition phase from a long-segment metallic rod construct to the nonfused thoracic spine. MethodsPure moment range of motion (ROM) tests (7.5 Nm) were performed on 7 cadaveric spine segments followed by compression (200 N). Specimens were tested in the following conditions: (1) intact; (2) T10-pelvis pedicle screws and rods (PSRs); and (3) extending the proximal construct to T6 using PEEK rods (PSR+PEEK). T10–11 rod strain, T9 anterolateral bone strain, and T10 screw bending moments were analyzed. ResultsAt the upper instrumented vertebra (UIV)+1, PSR+PEEK versus PSR significantly decreased ROM in flexion (115%, p=0.02), extension (104%, p=0.003), left lateral bending (46%, p=0.02), and right lateral bending (63%, p=0.008). Also, at UIV+1, PSR+PEEK versus intact significantly decreased ROM in flexion (111%, p=0.01) and extension (105%, p=0.003). The UIV+1 anterior column bone strain was significantly reduced with PSR+PEEK versus PSR during right lateral bending (p=0.02). Rod strain polarities reversed with PEEK rods in all loading directions except compression. ConclusionExtending a long-segment construct using PEEK rods caused a decrease in adjacent-level hypermobility as a consequence of long-segment immobilization and also redistributed the strain on the UIV and adjacent levels, which might contribute to PJK physiopathology. Further studies are necessary to observe the clinical outcomes of this technique.

Improving peptide-protein docking with AlphaFold-Multimer using forced sampling.

Protein interactions are key in vital biological processes. In many cases, particularly in regulation, this interaction is between a protein and a shorter peptide fragment. Such peptides are often part of larger disordered regions in other proteins. The flexible nature of peptides enables the rapid yet specific regulation of important functions in cells, such as their life cycle. Consequently, knowledge of the molecular details of peptide-protein interactions is crucial for understanding and altering their function, and many specialized computational methods have been developed to study them. The recent release of AlphaFold and AlphaFold-Multimer has led to a leap in accuracy for the computational modeling of proteins. In this study, the ability of AlphaFold to predict which peptides and proteins interact, as well as its accuracy in modeling the resulting interaction complexes, are benchmarked against established methods. We find that AlphaFold-Multimer predicts the structure of peptide-protein complexes with acceptable or better quality (DockQ ≥0.23) for 66 of the 112 complexes investigated—25 of which were high quality (DockQ ≥0.8). This is a massive improvement on previous methods with 23 or 47 acceptable models and only four or eight high quality models, when using energy-based docking or interaction templates, respectively. In addition, AlphaFold-Multimer can be used to predict whether a peptide and a protein will interact. At 1% false positives, AlphaFold-Multimer found 26% of the possible interactions with a precision of 85%, the best among the methods benchmarked. However, the most interesting result is the possibility of improving AlphaFold by randomly perturbing the neural network weights to force the network to sample more of the conformational space. This increases the number of acceptable models from 66 to 75 and improves the median DockQ from 0.47 to 0.55 (17%) for first ranked models. The best possible DockQ improves from 0.58 to 0.72 (24%), indicating that selecting the best possible model is still a challenge. This scheme of generating more structures with AlphaFold should be generally useful for many applications involving multiple states, flexible regions, and disorder.

Biochemical characterisation of glycosylated and deglycosylated forms of phytase from Cronobacter turicensis expressed in Pichia pastoris

Phytases are valuable industrial enzymes widely used in animal feed production, and when expressed in yeast, they undergo glycosylation. Herein, heterologous expression in Pichia pastoris and biochemical characterisation of glycosylated and deglycosylated forms of a novel phytase from Cronobacter turicensis belonging to the histidine acid phosphatase family were successfully carried out. Mutants with deleted N-glycosylation sites (N136, N171, and N202) were constructed by site-directed mutagenesis and characterised. Deglycosylation greatly changed the resistance of phytase to proteolysis, acidic pH, and temperature, but did not significantly affect other biochemical properties. High specific activity (1705 U/mg), Km (173 μM), and Vmax (2778 μmol min−1 mg−1), as well as the optimum temperature (50 °C) and pH (4.5) of the enzyme did not depend on the degree of glycosylation. The degree of change in resistance of phytase depended on the position of the N-glycosylation site on the protein globule. The removal of glycan at the N202 site located in the highly flexible region of the protein had the greatest effect on the enzyme characteristics and led to a decrease in resistance of phytase to pepsin and trypsin by 73% and 87%, respectively. Conversely, the glycosylated enzyme exhibited strong resistance to pepsin, trypsin, and acidic pH, and increased thermostability. It has been shown that N-glycosylation occurring in P. pastoris served as a powerful tool for improving the biochemical properties of recombinant phytase and made it promising for use as an animal feed additive.

Natural polyphenols convert proteins into histone-binding ligands

Antioxidants are sensitive to oxidation and are immediately converted into their oxidized forms that can react with proteins. We have recently found that proteins incubated with oxidized vitamin C (dehydroascorbate) gain a new function as a histone-binding ligand. This finding led us to predict that antioxidants, through conversion to their oxidized forms, may generally have similar functions. In the present study, we identified several natural polyphenols as a source of histone ligands and characterized the mechanism for the interaction of protein-bound polyphenols with histone. Through screening of 25 plant-derived polyphenols by assessing their ability to convert bovine serum albumin into histone ligands, we identified seven polyphenols, including (-)-epigallocatechin-3-O-gallate (EGCG). Additionally, we found that the histone tail domain, which is a highly charged and conformationally flexible region, is involved in the interaction with the polyphenol-modified proteins. Further mechanistic studies showed the involvement of a complex heterogeneous group of the polyphenol-derived compounds bound to proteins as histone-binding elements. We also determined that the interaction of polyphenol-modified proteins with histones formed aggregates and exerted a protective effect against histone-mediated cytotoxicity toward endothelial cells. These findings demonstrated that histones are one of the major targets of polyphenol-modified proteins and provide important insights into the chemoprotective functions of dietary polyphenols.

Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

The Role of Disordered Regions in Orchestrating the Properties of Multidomain Proteins: The SARS-CoV-2 Nucleocapsid Protein and Its Interaction with Enoxaparin.

Novel and efficient strategies need to be developed to interfere with the SARS-CoV-2 virus. One of the most promising pharmaceutical targets is the nucleocapsid protein (N), responsible for genomic RNA packaging. N is composed of two folded domains and three intrinsically disordered regions (IDRs). The globular RNA binding domain (NTD) and the tethered IDRs are rich in positively charged residues. The study of the interaction of N with polyanions can thus help to elucidate one of the key driving forces responsible for its function, i.e., electrostatics. Heparin, one of the most negatively charged natural polyanions, has been used to contrast serious cases of COVID-19 infection, and we decided to study its interaction with N at the molecular level. We focused on the NTR construct, which comprises the NTD and two flanking IDRs, and on the NTD construct in isolation. We characterized this interaction using different nuclear magnetic resonance approaches and isothermal titration calorimetry. With these tools, we were able to identify an extended surface of NTD involved in the interaction. Moreover, we assessed the importance of the IDRs in increasing the affinity for heparin, highlighting how different tracts of these flexible regions modulate the interaction.

Topoisomerase Enzyme Inhibitors as Potential Drugs Against Cancer: What Makes Them Selective or Dual? - A Review.

Topoisomerase inhibitors are extensively used in cancer chemotherapy. In the process of identifying novel anticancer compounds, biological evaluations are crucial and include, among others, the use of in silico and in vitro approaches. This work aimed to present recent research involving the obtainment and in silico and in vitro evaluation of topoisomerase I, II, and double inhibitors, of synthetic and natural origin, as potential compounds against tumor cells, in addition to proposing the construction of a desirable enzyme catalytic site. Therefore, it was observed that most Topoisomerase I inhibitors presented medium to large structures, with a rigid portion and a flexible region. In contrast, Topoisomerase IIα inhibitors showed medium and large structural characteristics, in addition to the planarity of the aromatic rings, which are mitigated due to flexible rings but may also present elements that restrict conformation. Most compounds that exhibit dual inhibitory activity had relatively long chains, in addition to a flat and rigid portion suggestive of affinity for Topo I and a flexible region characteristic of selective drugs for Topo II. Besides, it is noticed that most compounds that exhibit dual inhibitory showed similarities in the types of interactions and amino acids when compared to the selective compounds of Topo I and II. For instance, selective Topoisomerase I inhibitors interact with Arginine364 residues, and selective Topoisomerase II inhibitors interact with Arginine487 residues, as both residues are targets for dual compounds.

Polytrauma patient with lateral thoracic spondyloptosis: case report and literature review

Background: Spondyloptosis is caused by high force trauma. The vast majority of cases occur in the sagittal plane and at transition points where ridged sections meet more flexible regions. Lateral thoracic spondyloptosis is extremely rare and there is no current consensus on the optimal treatment plan.

Improving Thermostability of GH11 Xylanase XynASP by the Design of Loop Region

Highly flexible loop regions can affect the structure-function relationship of enzymes. In this study, to reveal the effect of the loop on the thermal stability of GH11 xylanase XynASP from Aspergillus saccharolyticus JOP 1030-1, three mutants (T41V, A79Y, T81Q) located in the loop region were predicted by the FireProt web server and constructed by site-directed mutagenesis. Heat tolerance experiments were performed in the mutants and wild-type XynASP, as well as for previously reported mutant T41W, indicating that the thermostability of enzymes from weak to strong was wild-type XynASP, T41V, T81Q, A79Y and T41W. Novel hydrophobic contacts and hydrogen bonds in the single mutants were found and explained for enhanced thermostability. T41W, A79Y and T81Q were combined by site-directed mutagenesis. The combined double mutants T41W/A79Y and A79Y/T81Q displayed significantly higher thermostability than that of the wild-type, yet lower than that of the robust mutant T41W after 30 min of incubation at 35–60 °C. But the triple mutant T41W/A79Y/T81Q displayed a slight improvement in thermal stability compared to T41W after 30 min of incubation at 35–60 °C. Meanwhile, T41W/A79Y/T81Q exhibited substantially improved thermostability with a half-life of inactivation enhanced from 20.7 min to 127.0 min at 45 °C. Furthermore, the optimum temperature increased by 10 °C compared to the wild-type XynASP. The pH stability of T41W/A79Y/T81Q at pH 3.0-8.0 all obtained more than 88% residual activities, displaying a significant improvement compared to the wild-type XynASP (40–70% residual activities) at the same conditions. This study confirms that designing GH11xylanases in the loop region is an effective strategy for obtaining thermostabilizing xylanases.

Fibrous anisotropy and mineral gradients within the radula stylus of chiton: Controlled stiffness and damage tolerance in a flexible biological composite

Over hundreds of millions of years, organisms have evolved architected structures via precise control over hierarchically assembled components, including the integration of dissimilar materials. One such example is found in the radula system of chitons, intertidal mollusks that feed on algae growing on the rock. Their radula consists of multiple rows of ultrahard teeth, each integrated with a foldable belt-like substrate via a stiff, yet flexible stylus, which is essential for efficient rasping during the feeding process. Here, we investigate the nano and micro-scale components and architectures as well as regional mechanical properties of the stylus, and their subsequent role during the rasping of Cryptochiton stelleri. Three important factors were determined to contribute to the regio-specific stiffness of the stylus: the presence of mineral components, highly oriented chitinous fibers, and a chemically cross-linked protein matrix. All these factors are varied throughout the stylus. There is a high mineral content on the trailing edge close to the tooth and a cross-linked matrix on the leading edge, both with orientational specific oriented chitin fibers that provide force transduction to the tooth. Conversely, there is a significant lack of mineral or cross-linked matrix in the proximal end as well as a low degree of fiber orientation, resulting in a flexible region that can accommodate torsion and flexure during rasping. Understanding the graded composite structure of the stylus and applying this unique design to various engineering fields such as soft robotics, biotechnology, and the medical industry, can inspire the production of high-performance materials.

Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P.

Chromatin marks are recognized by distinct binding modules, many of which are embedded in multidomain proteins. How the different functionalities of such complex chromatin modulators are regulated is often unclear. Here, we delineated the interplay of the H3 amino terminus– and K9me-binding activities of the multidomain hUHRF1 protein. We show that the phosphoinositide PI5P interacts simultaneously with two distant flexible linker regions connecting distinct domains of hUHRF1. The binding is dependent on both, the polar head group, and the acyl part of the phospholipid and induces a conformational rearrangement juxtaposing the H3 amino terminus and K9me3 recognition modules of the protein. In consequence, the two features of the H3 tail are bound in a multivalent, synergistic manner. Our work highlights a previously unidentified molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

Integrative model of the FSH receptor reveals the structural role of the flexible hinge region

The follicle-stimulating hormone receptor (FSHR) belongs to the glycoprotein hormone receptors, a subfamily of G-protein-coupled receptors (GPCRs). FSHR is involved in reproductive processes such as gonadal development and maturation. Structurally, the extensive extracellular domain, which contains the hormone-binding site and is linked to the transmembrane domain by the hinge region (HR), is characteristic for these receptors. How this HR is involved in hormone binding and signal transduction is still an open question. We combined invitro and in situ chemical crosslinking, disulfide pattern analysis, and mutation data with molecular modeling to generate experimentally driven full-length models. These models provide insights into the interface, important side-chain interactions, and activation mechanism. The interface indicates a strong involvement of the connecting loop. A major rearrangement of the HR seems implausible due to the tight arrangement and fixation by disulfide bonds. The models are expected to allow for testable hypotheses about signal transduction and drug development for GPHRs.

Increasing displacement range in 3D printed compliant joints via bio-inspired slot patterns: An exploratory study

Compliant joints own their motion performance to three main aspects: base material, geometrical design, and dimensions. When aiming to increase their motion range without affecting the overall assembly of the system in which they are employed, modifying the design or dimensions is not appropriate anymore. To overcome this, the current work presents a novel strategy for increasing the motion range of the split tube flexural pivots joint based on removing material from the complaint joint using two specific bio-inspired patterns. These bio-inspired patterns are based on the shells encountered in Testudines (tortoise) and Dasypodidae (armadillo). Then, these patterns were parametrized, and their effective mechanical properties evaluated on 3D printed samples. Features of the flexible region of the flexural pivot joint were varied to identify the conditions in which the motion range increases. A factorial analysis was made to obtain the angular displacement response based on the determined parameters, which depend on the flexible region features. The use of these patterns successfully demonstrated the reduction in stiffness of the joints, obtaining increments in displacements twice in the stiffest case and 4.25 times in the most flexible one. Secondary displacements were also analyzed. Displacements measured were characterized via computationally analyzed images and recordings. Finally, the mechanical properties measured were compared to computationally predicted properties via Finite Element Models. Predictions resulted in good agreement with the experimental measurements with errors below 10% in most cases and up to 15% in the worst cases. These were attributed to manufacturing defects in the samples.

Improved thermostability of lipase Lip2 from Yarrowia lipolytica through disulfide bond design for preparation of medium-long-medium structured lipids

Lipase Lip2 from Yarrowia lipolytica with great potential value in the enzymatic preparation of medium-long-medium structured lipids (MLM-SLs), but the synthetic efficiency might be limited by its poor thermostability. We identified flexible regions of lipase Lip2 by molecular dynamics (MD) simulation, and then disulfide bonds were engineered into these regions to enhance its thermostability. Compared with the wild type, mutant 4sN with four disulfide bonds showed a 19.22 and 27.75 °C increase in the melting temperature (Tm) and the half-loss temperature at 15 min (T15 50), respectively. The half-life of the wild type at 50 °C was 1.66 min, while that of mutant 4sN at 60 °C was 40.52 min. MD simulation showed that the thermal stability improvement in the mutant was attributed to enhanced structural rigidity. Finally, the MLM-SLs were synthesized from olive oil and caprylic acid with immobilized wild-type and mutant 4sN. Mutant 4sN displayed a 5 °C higher optimum temperature and a higher reaction rate than the wild type. Meanwhile, residual activity of mutant 4sN after 7 consecutive cycles increased by 20.84% compared to the wild type. The increased reaction rate and recyclability of lipase Lip2 provide greater potential for industrial production of MLM-SLs.

Generation of Reporter-Expressing New World Arenaviruses: A Systematic Comparison.

Replication-competent reporter-expressing viruses are crucial tools in molecular virology with applications that range from antiviral screening to live-cell imaging of protein spatiotemporal dynamics. However, there is currently little information available regarding viable strategies to develop reporter-expressing arenaviruses. To address this, we used Tacaribe virus (TCRV), an apathogenic BSL2 arenavirus, to assess the feasibility of different reporter expression approaches. We first generated trisegmented TCRV viruses with either the glycoprotein (GP) or nucleoprotein (NP) replaced by a reporter (GFP, mCherry, or nanoluciferase). These viruses were all viable, but showed marked differences in brightness and attenuation. Next, we generated terminal fusions with each of the TCRV proteins (i.e., NP, GP, polymerase (L), matrix protein (Z)) either with or without a T2A self-cleavage site. We tested both the function of the reporter-fused proteins alone, and the viability of corresponding recombinant TCRVs. We successfully rescued viruses with both direct and cleavable reporter fusions at the C-terminus of Z, as well as cleavable N-terminal fusions with NP. These viruses all displayed detectable reporter activity, but were also moderately attenuated. Finally, reporter proteins were inserted into a flexible hinge region within L. These viruses were also viable and showed moderate attenuation; however, reporter expression was only detectable for the luminescent virus. These strategies provide an exciting range of new tools for research into the molecular biology of TCRV that can likely also be adapted to other arenaviruses.

Identification of the initial reactive sites of micellar and non-micellar casein exposed to microbial transglutaminase

To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αs1 K34–αs2 Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins.Graphical abstract

In silico investigation of Alsin RLD conformational dynamics and phosphoinositides binding mechanism.

Alsin is a protein known for its major role in neuronal homeostasis and whose mutation is associated with early-onset neurodegenerative diseases. It has been shown that its relocalization from the cytoplasm to the cell membrane is crucial to induce early endosomes maturation. In particular, evidences suggest that the N-terminal regulator of chromosome condensation 1 like domain (RLD) is necessary for membrane association thanks to its affinity to phosphoinositides, membrane lipids involved in the regulation of several signaling processes. Interestingly, this domain showed affinity towards phosphatidylinositol 3-phosphate [PI(3)P], which is highly expressed in endosomes membrane. However, Alsin structure has not been experimentally resolved yet and molecular mechanisms associated with its biological functions are mostly unknown. In this work, Alsin RLD has been investigated through computational molecular modeling techniques to analyze its conformational dynamics and obtain a representative 3D model of this domain. Moreover, a putative phosphoinositide binding site has been proposed and PI(3)P interaction mechanism studied. Results highlight the substantial conformational stability of Alsin RLD secondary structure and suggest the role of one highly flexible region in the phosphoinositides selectivity of this domain.