Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (NSpaA) that is also crosslinked by CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.

Growing interest in i-motif DNA as a transcriptional regulatory element motivates development of synthetic molecules capable of targeting these structures. In this study, we designed unmodified peptide nucleic acid (PNA) and gamma-modified PNA (γPNA) oligomers complementary to an i-motif forming sequence derived from the promoter of the KRAS oncogene. Biophysical techniques such as circular dichroism (CD) spectroscopy, CD melting, and fluorescence spectroscopy demonstrated the successful invasion of the i-motif by PNA and γPNA. Both PNA and γPNA showed very strong binding to the target sequence with high thermal stability of the resulting heteroduplexes. Interestingly fluorescence and CD experiments indicated formation of an intermolecular i-motif structure via the overhangs of target-probe heteroduplexes formed by PNA/γPNA invasion of the intramolecular i-motif. Targeting promoter i-motif forming sequences with high-affinity oligonucleotide mimics like γPNAs may represent a new approach for inhibiting KRAS transcription, thereby representing a potentially useful anti-cancer strategy.

DJ-1 is a homodimeric protein that is centrally involved in various human diseases including Parkinson disease (PD). DJ-1 protects against oxidative damage and mitochondrial dysfunction through a homeostatic control of reactive oxygen species (ROS). DJ-1 pathology results from a loss of function, where ROS readily oxidizes a highly conserved and functionally essential cysteine (C106). The over-oxidation of DJ-1 C106 leads to a dynamically destabilized and biologically inactivated protein. An analysis of the structural stability of DJ-1 as a function of oxidative state and temperature may provide further insights into the role the protein plays in PD progression. NMR spectroscopy, circular dichroism, analytical ultracentrifugation sedimentation equilibrium, and molecular dynamics simulations were utilized to investigate the structure and dynamics of the reduced, oxidized (C106-SO2−), and over-oxidized (C106-SO3−) forms of DJ-1 for temperatures ranging from 5°C to 37°C. The three oxidative states of DJ-1 exhibited distinct temperature-dependent structural changes. A cold-induced aggregation occurred for the three DJ-1 oxidative states by 5°C, where the over-oxidized state aggregated at significantly higher temperatures than both the oxidized and reduced forms. Only the oxidized and over-oxidized forms of DJ-1 exhibited a mix state containing both folded and partially denatured protein that likely preserved secondary structure content. The relative amount of this denatured form of DJ-1 increased as the temperature was lowered, consistent with a cold-denaturation. Notably, the cold-induced aggregation and denaturation for the DJ-1 oxidative states were completely reversible. The dramatic changes in the structural stability of DJ-1 as a function of oxidative state and temperature are relevant to its role in PD and its functional response to oxidative stress.

Translocation of positively charged cell penetrating peptides (CPP) through cell membrane is important in drug delivery. Here we report all-atom molecular dynamics simulations to investigate how a biphosphate salt in a solvent affects the interaction of a CPP, HIV-1 Tat peptide with model dipalmitoylphosphatidylcholine (DPPC) lipid bilayer. Tat peptide has a large number of basic arginines and a couple of polar glutamines. We observe that in absence of salt, the basic residues of the polypeptide get localized in the vicinity of the membrane without altering the bilayer properties much; polypeptide induce local thinning of the bilayer membrane at the area of localization. In presence of biphosphate salt, the basic residues, dressed by the biphosphate ions, are repelled by the phosphate head groups of the lipid molecules. However, polar glutamine prefers to stay in the vicinity of the bilayer. This leads to larger local bilayer thickness at the contact point by the polar residue and non-uniform bilayer thickness profile. The thickness deformation of bilayer structure disappears upon mutating the polar residue, suggesting importance of the polar residue in bilayer deformation. Our studies point to control bilayer deformation by appropriate peptide sequence and solvent conditions.

The Panspermia hypothesis posits that either life's building blocks (molecular Panspermia) or life itself (organism-based Panspermia) may have been interplanetarily transferred to facilitate the origins of life (OoL) on a given planet, complementing several current OoL frameworks. Although many spaceflight experiments were performed in the past to test for potential terrestrial organisms as Panspermia seeds, it is uncertain whether such organisms will likely “seed” a new planet even if they are able to survive spaceflight. Therefore, rather than using organisms, using abiotic chemicals as seeds has been proposed as part of the molecular Panspermia hypothesis. Here, as an extension of this hypothesis, we introduce and review the plausibility of a polymeric material-based Panspermia seed (M-BPS) as a theoretical concept, where the type of polymeric material that can function as a M-BPS must be able to: (1) survive spaceflight and (2) “function”, i.e., contingently drive chemical evolution toward some form of abiogenesis once arriving on a foreign planet. We use polymeric gels as a model example of a potential M-BPS. Polymeric gels that can be prebiotically synthesized on one planet (such as polyester gels) could be transferred to another planet via meteoritic transfer, where upon landing on a liquid bearing planet, can assemble into structures containing cellular-like characteristics and functionalities. Such features presupposed that these gels can assemble into compartments through phase separation to accomplish relevant functions such as encapsulation of primitive metabolic, genetic and catalytic materials, exchange of these materials, motion, coalescence, and evolution. All of these functions can result in the gels' capability to alter local geochemical niches on other planets, thereby allowing chemical evolution to lead to OoL events.

G-quadruplexes (G4s), the noncanonical nucleic acid secondary structure, form within guanine-rich DNA or RNA sequences. G4s formation can affect chromatin architecture and gene regulation and has been associated with various cellular functions, including DNA replication, transcription, and genome maintenance. Visualizing and detecting G4s precisely in such processes is essential to increasing our understanding of G4s biology. Considerable attention has focused on the G4s targeting molecular imaging studies. Besides, fluorescent light-up aptamers (FLAPs, also referred to as fluorogenic aptamers) have gained momentum, which commonly have a G4 scaffolding for imaging intracellular RNAs and metabolites. In this review, we first introduce several representative fluorescent imaging approaches for tracking G4s in cells and in vivo. We also discuss the potential of G4-containing FLAPs in bioimaging and summarize current developments in this field from the standpoint of fluorescent molecules. Finally, we discuss the present challenges and future potential of G4 imaging and G4-containing FLAPs development.

The biological functions of polysaccharides are influenced by their chemistry and chain conformation, which have resulted in various functional applications and new uses for polysaccharides in recent years. Sacran is an intriguing ampholytic polysaccharide with several key properties such as metal adsorption, anti-inflammatory nature, and transdermal drug-carrying capacity. It has an extremely high molecular weight over 107 g/mol, which is much higher than those of the previously reported microbial polysaccharides. In particular, it has a remarkable self-orienting characteristic over a large length scale, which could produce a bundle with twisted morphologies from the nanoscale to the microscale with diameters of ~1 μm and lengths of >800 μm. In this review, morphological variations, as well as novel self-organization and hierarchical self-assembly are comprehensively discussed. Sacran fibers deform into various forms, such as two- and three-dimensional flexible fibers and micro–nano fragments, during their evaporation. The self-assembly and disassembly of the sacran are explained in terms of the preparation process and factors that influence the morphology. This review will pave the way for the development of novel modules such as humidity-sensitive actuators, micro-patterned cell scaffolds, and uniaxially oriented membranes.

Polymeric barrier membranes are used in periodontal applications to prevent fibroblastic cell migration into the cavities of bone tissue and to properly guide the proliferation of tissues. In this study, the fabrication, characterization, bioactivity, and in vitro biological properties of polyvinyl alcohol-based nanofibrous membranes containing nano-sized 45S5 bioactive glass (BG) loaded with chlorhexidine (CH) gluconate with biocompatible, bioactive, and antibacterial properties for using as dental barrier membranes were investigated. Nanofibrous membranes with an average fiber diameter, pore size, and porosity of 210 nm, 24.73 μm, and 12.42%, respectively, were loaded with 1% and 2% CH, and the release profile was investigated. The presence of BG in the membranes promoted fibroblastic proliferation and the presence of CH provided antibacterial properties. Nanofibrous membranes exhibit a high ability to restrict bacterial growth while fulfilling the necessary conditions for use as a dental barrier thanks to their low swelling rates, significant surface bioactivities, and appropriate degradation levels.

In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing – comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.

The simplest way to account for the influence of diffusion on the kinetics of multisite phosphorylation is to modify the rate constants in the conventional rate equations of chemical kinetics. We have previously shown that this is not enough and new transitions between the reactants must also be introduced. Here we extend our results by considering enzymes that are inactive after modifying the substrate and need time to become active again. This generalization leads to a surprising result. The introduction of enzyme reactivation results in a diffusion-modified kinetic scheme with a new transition that has a negative rate constant. The reason for this is that mapping non-Markovian rate equations onto Markovian ones with time-independent rate constants is not a good approximation at short times. We then developed a non-Markovian theory that involves memory kernels instead of rate constants. This theory is now valid at short times, but is more challenging to use. As an example, the diffusion-modified kinetic scheme with new connections was used to calculate kinetics of double phosphorylation and steady-state response in a phosphorylation-dephosphorylation cycle. We have reproduced the loss of bistability in the phosphorylation-dephosphorylation cycle when the enzyme reactivation time decreases, which was obtained by particle-based computer simulations.

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.

Plastic materials for food packaging are being replaced by biodegradable films based on biopolymers due to the adverse effects they have had on animal life and the environment. In this study, nanocomposite films containing 2.5 wt% sodium caseinate and 2 wt% glycerol were reinforced with 0.1 or 0.2 wt% nano TiO2 prepared in two forms: spheres (P25) and tubes. The effects of nanoreinforcement geometry on mechanical, tensile, barrier, thermogravimetric, and optical properties, and distribution of nanoparticles were described. The interactions among film components were analyzed by Fourier transform infrared spectroscopy (FTIR). Addition of nanotubes significantly increased E' (341 wt%) and E" (395 wt%) moduli, the Young modulus E (660 wt%), the residual mass at 500°C (38 wt%), and color change (6.78) compared to control film. The compositional mapping studies showed that P25 nanoparticles were homogeneously distributed between the surfaces of the film while nanotubes were found on the bottom surface. The changes in position of the FTIR spectra signals as compared to pure protein signals indicated strong matrix/reinforcement interactions. In addition, the changes in intensity in 1100, 1033, and 1638 cm−1 FTIR signals suggested formation of a protein/Tween 20 ester. The geometry of reinforcement was highly relevant regarding physical properties, showing nanotubes as being very successful for enhancing tensile properties.

Over the past 3.5 billion years of evolution, enzymes have adopted a myriad of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be disallowed to all amino acids, except glycine, due to steric hindrance. Triosephosphate isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site lysine residue (K13) with dihedrals within the fourth quadrant (Φ = +51/Ψ = −143). Both the amino acid and the dihedral angles are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β-sheet dihedral values of Φ = -135/Ψ = +170 but experiments show a 1000-fold loss in activity. Based on these results, we hypothesized that adopting the unfavorable torsion angle for K13 contributes to catalysis. Using both, computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine. In silico molecular dynamics (MD) simulations were performed using 2JK2 unliganded human TIM as a starting structure. Ramachandran plots, containing K13 dihedral values reveal full or partial loss of disallowed zone angles. N11A showed no detectable catalytic activity and lost the unfavorable K13 dihedral angles across four separate force fields during simulation while all other mutants plus wild type retained activity and retained the conserved K13 dihedral angles.

Human papillomavirus (HPV) is recognized as the causative agent of cervical cancer in women, and it is associated with other anogenital and head/neck cancers. More than 120 types of HPV have been identified and many classified as high- or low-risk according to their oncogenic potential. One of its proteins, E6, has evolved to overcome the oncosuppressor functions of p53 by targeting this protein for degradation via interaction with the human ubiquitin-ligase E6AP. This study evaluates the correlation between the association strength of 40 HPV E6 types to the E6AP/p53 complex and the HPV oncogenesis risk using molecular simulations and machine and deep learning (ML/DL). In addition, a ML/DL-driven prediction is proposed for the HPV unclassified oncogenic risk type. The results indicate that thermodynamics play a pivotal role in the establishment of HPV-associated cancer and highlight the need to include some viral types in the HPV-related cancer surveillance and prevention strategies.

Specific recognition of DNA base sequences by proteins is vital for life-cycles of all organisms. In a large number of crystal structures of protein–DNA complexes, DNA conformation significantly deviates from the canonical B-DNA structure. A key question is whether such alternate conformations exist prior to protein binding and one is selected for complexation or the structure observed is induced by protein binding. Non-canonical base pairs, such as Hoogsteen base pairs, are often observed in crystal structures of protein–DNA complexes. We decided to explore whether the occurrence of such non-canonical base pairs in protein–DNA complexes is induced by the protein or is selected from pre-existing conformations. Detailed quantum chemical calculations with dispersion-corrected density functional theory (DFT-D) indicated that most of the non-canonical base pairs with DNA bases are stable even in the absence of the interacting amino acids. However, the G:G Hoogsteen base pair, which also appears in the telomere structure, appears to be unstable in the absence of other stabilizing agents, such as positively charged amino acids. Thus, the stability of many of the non-canonical base pair containing duplexes may be close to the canonical B-DNA structure and hence energetically accessible in the ground state; suggesting that the selection from pre-existing conformations may be an important mechanism for observed non-canonical base pairs in protein–DNA complexes.

Before reaching their targets, sequence-specific DNA-binding proteins nonspecifically bind to DNA through electrostatic interactions and stochastically change their locations on DNA. Investigations into the dynamics of DNA-scanning by proteins are nontrivial due to the simultaneous presence of multiple translocation mechanisms and many sites for the protein to nonspecifically bind to DNA. Nuclear magnetic resonance (NMR) spectroscopy can provide information about the target DNA search processes at an atomic level. Paramagnetic relaxation enhancement (PRE) is particularly useful to study how the proteins scan DNA in the search process. Previously, relatively simple two-state or three-state exchange models were used to explain PRE data reflecting the target search process. In this work, using more realistic discrete-state stochastic kinetics models embedded into an NMR master equation, we analyzed the PRE data for the HoxD9 homeodomain interacting with DNA. The kinetic models that incorporate sliding, dissociation, association, and intersegment transfer can reproduce the PRE profiles observed at some different ionic strengths. The analysis confirms the previous interpretation of the PRE data and shows that the protein's probability distribution among nonspecific sites is nonuniform during the target DNA search process.

Coevolution between protein residues is normally interpreted as direct contact. However, the evolutionary record of a protein sequence contains rich information that may include long-range functional couplings, couplings that report on homo-oligomeric states or even conformational changes. Due to the complexity of the sequence space and the lack of structural information on various members of a protein family, it has been difficult to effectively mine the additional information encoded in a multiple sequence alignment (MSA). Here, taking advantage of the recent release of the AlphaFold (AF) database we attempt to identify coevolutionary couplings that cannot be explained simply by spatial proximity. We propose a simple computational method that performs direct coupling analysis on a MSA and searches for couplings that are not satisfied in any of the AF models of members of the identified protein family. Application of this method on 2012 protein families suggests that ~12% of the total identified coevolving residue pairs are spatially distant and more likely to be disordered than their contacting counterparts. We expect that this analysis will help improve the quality of coevolutionary distance restraints used for structure determination and will be useful in identifying potentially functional/allosteric cross-talk between distant residues.

Elastin-like peptides (ELPs) are thermoresponsive biopolymers inspired by the characteristic repetitive sequences of natural elastin. As ELPs exhibit temperature-dependent reversible self-assembly, they are expected to be biocompatible thermoresponsive materials for drug delivery carriers. One of the most widely studied ELPs in this field is the repetitive pentapeptide, (VPGXG)n. We previously reported that phenylalanine-containing ELP (Fn) analogs, in which the former Val residue of the repetitive sequence (VPGVG)n is replaced by Phe, show coacervation with a short chain length (n = 5). Owing to their short sequences, Fn analogs are easily modified in amino acid sequences via simple chemical synthesis, and are useful for investigating the relationship between peptide sequences and temperature responsiveness. In this study, we developed Fn analogs by replacing Phe residue(s) with other amino acids or introducing another amino acid at the N-terminus. The temperature responsiveness of the Fn analogs changed drastically with the substitution of a single Phe residue, suggesting that aromatic amino acids play an important role in their self-assembly. In addition, the self-assembling ability of Fn was enhanced by increasing the bulkiness of the N-terminal amino acids. Therefore, the N-terminal residue was considered to be important for hydrophobicity-induced intermolecular interactions between the peptides during coacervation.

Two nanoformulations with mucoadhesive properties and brain-targeting mechanisms were designed to deliver the anti-Parkinson's drug, ropinirole hydrochloride (RH). In the first formulation, RH and the amphiphilic block copolymer methoxy poly(ethylene glycol)-b-poly(caprolactone) were assembled in a core–shell morphology followed by coating with a mucoadhesive chitosan outer layer producing a multilayer vehicle (MLV). In the second formulation, RH was encapsulated during the polyelectrolyte complexation of two natural polymers, chitosan and alginate producing RH-loaded chitosan-alginate polyelectrolyte (PEC) nanocomplex. Conditions of each formulation were adopted for optimal drug loading. Physico-chemical characterization of the prepared formulations (particle size, polydispersity index and zeta-potential) exhibited stable monodispersed nanoparticles. RH was radiolabeled by I-131 radiotracer in a high-radiochemical yield. Biodistribution and brain targeting of RH from the prepared formulations were studied after administration of 131I-RH-loaded nanoparticles to albino mice via intranasal and intravenous routs. Elevated brain radioactivity was detected post IN administration of (131I-RH/PCL-PEG/CS) nanoparticles and (131I-RH/CS-ALG) nanoparticles comparing with the IN administrated RH solutions (Cmax = 2.8 ± 0.3, 2 ± 0.3, 0.93 ± 0.03% radioactivity/g, 1 h post administration, respectively). This demonstrated that a relatively high-brain targeting could be achieved via intranasal route of administration of RH-loaded nanoparticles. The proposed models are further potential for application to deliver many other brain-targeting therapeutics.

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL−1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.