Triplet-Triplet Annihilation Upconverting Liposomes: Mechanistic Insights into the Role of Membranes in Two-Dimensional TTA-UC.

Triplet-triplet annihilation upconversion (TTA-UC) implemented in nanoparticle assemblies is of emerging interest in biomedical applications, including in drug delivery and imaging. As it is a bimolecular process, ensuring sufficient mobility of the sensitizer and annihilator to facilitate effective collision in the nanoparticle is key. Liposomes can provide the benefits of two-dimensional confinement and condensed concentration of the sensitizer and annihilator along with superior fluidity compared to other nanoparticle assemblies. They are also biocompatible and widely applied across drug delivery modalities. However, there are relatively few liposomal TTA-UC systems reported to date, so systematic studies of the influence of the liposomal environment on TTA-UC are currently lacking. Here, we report the first example of a BODIPY-based sensitizer TTA-UC system within liposomes and use this system to study TTA-UC generation and compare the relative intensity of the anti-Stokes signal for this system as a function of liposome composition and membrane fluidity. We report for the first time on time-resolved spectroscopic studies of TTA-UC in membranes. Nanosecond transient absorption data reveal the BODIPY-perylene dyad sensitizer has a long triplet lifetime in liposome with contributions from three triplet excited states, whose lifetimes are reduced upon coinclusion of the annihilator due to triplet-triplet energy transfer, to a greater extent than in solution. This indicates triplet energy transfer between the sensitizer and the annihilator is enhanced in the membrane system. Molecular dynamics simulations of the sensitizer and annihilator TTA collision complex are modeled in the membrane and confirm the co-orientation of the pair within the membrane structure and that the persistence time of the bound complex exceeds the TTA kinetics. Modeling also reliably predicted the diffusion coefficient for the sensitizer which matches closely with the experimental values from fluorescence correlation spectroscopy. The relative intensity of the TTA-UC output across nine liposomal systems of different lipid compositions was explored to examine the influence of membrane viscosity on upconversion (UC). UC showed the highest relative intensity for the most fluidic membranes and the weakest intensity for highly viscous membrane compositions, including a phase separation membrane. Overall, our study reveals that the co-orientation of the UC pair within the membrane is crucial for effective TTA-UC within a biomembrane and that the intensity of the TTA-UC output can be tuned in liposomal nanoparticles by modifying the phase and fluidity of the liposome. These new insights will aid in the design of liposomal TTA-UC systems for biomedical applications.

Screening Stability, Thermochemistry, and Chemical Kinetics of 3-Hydroxybutanoic Acid as a Bifunctional Biodiesel Additive.

The thermo-kinetic aspects of 3-hydroxybutyric acid (3-HBA) pyrolysis in the gas phase were investigated using density functional theory (DFT), specifically the M06-2X theoretical level in conjunction with the cc-pVTZ basis set. The obtained data were compared with benchmark CBS-QB3 results. The degradation mechanism was divided into 16 pathways, comprising 6 complex fissions and 10 barrierless reactions. Energy profiles were calculated and supplemented with computations of rate coefficients and branching ratios over the temperature range of 600-1700 K at a pressure of 1 bar using transition state theory (TST) and Rice-Ramsperger-Kassel-Marcus (RRKM) methods. Thermodynamics results indicated the presence of six stable conformers within a 4 kcal mol-1 energy range. The estimated chemical kinetics results suggested that TST and RRKM approaches are comparable, providing confidence in our calculations. The branching ratio analysis reveals that the dehydration reaction pathway leading to the formation of H2O and CH3CH═CHCO2H dominates entirely at T ≤ 650 K. At these temperatures, there is a minor contribution from the simple homolytic bond fission reaction, yielding related radicals [CH3•CHOH + •CH2CO2H]. However, at T ≥ 700 K, this reaction becomes the primary decomposition route. At T = 1700 K, there is a minor involvement of a reaction pathway resulting in the formation of CH3CH(OH)•CH2 + •CHO(OH) with an approximate contribution of 16%, and a reaction leading to [•CH3 + •CH2OHCH2CO2H] with around 9%.

Computational analysis of substituent effects on proton affinity and gas-phase basicity of TEMPO derivatives and their hydrogen bonding interactions with water molecules.

The study investigates the molecular structure of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its derivatives in the gas phase using B3LYP and M06-2X functional methods. Intermolecular interactions are analyzed using natural bond orbital (NBO) and atoms in molecules (AIM) techniques. NO2-substituted TEMPO displays high reactivity, less stability, and softer properties. The study reveals that the stability of TEMPO derivatives is mainly influenced by LP(e) → σ∗ electronic delocalization effects, with the highest stabilization observed on the oxygen atom of the nitroxide moiety. This work also considers electron density, atomic charges, and energetic and thermodynamic properties of the studied NO radicals, and their relative stability. The proton affinity and gas-phase basicity of the studied compounds were computed at T = 298 K for O-protonation and N-protonation, respectively. The studied DFT method calculations show that O-protonation is more stable than N-protonation, with an energy difference of 16.64-20.77 kcal/mol (22.80-25.68 kcal/mol) at the B3LYP (M06-2X) method. The AIM analysis reveals that the N-O…H interaction in H2O complexes has the most favorable hydrogen bond energy computed at bond critical points (3, - 1), and the planar configurations of TEMPO derivatives exhibit the highest EHB values. This indicates stronger hydrogen bonding interactions between the N-O group and water molecules.

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development.

The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.

Why Are Left-Handed G-Quadruplexes Scarce?

G-quadruplexes (G4s) are nucleic acid structures crucial for the regulation of gene expression and genome maintenance. While they hold promise as nanodevice components, achieving desired G4 folds requires understanding the interplay between stability and structural properties, like helicity. Although right-handed G4 structures dominate the experimental data, the molecular basis for this preference over left-handed helicity is unclear. To address this, we employ all-atom molecular dynamics simulations and quantum chemical methods. Our results reveal that right-handed G4s exhibit greater thermodynamic and kinetic stability as a result of favorable sugar-phosphate backbone conformations in guanine tracts. Moreover, while hydrogen-bonding patterns influence helicity-specific G4 loop conformations, they minimally affect stability differences. We also elucidate the strong correlation between helicity and the strand progression direction, essential for G4 structures. These findings deepen our understanding of G4s, providing molecular-level insights into their structural and energetic preferences, which could inform the design of novel nanodevices.

Low-Barrier Hydrogen Bond Determines Target-Binding Affinity and Specificity of the Antitubercular Drug Bedaquiline.

The role of short strong hydrogen bonds (SSHBs) in ligand-target binding remains largely unexplored, thereby hindering a potentially important avenue in rational drug design. Here we investigate the interaction between the antituberculosis drug bedaquiline (Bq) and the mycobacterial ATP synthase to unravel the role of a specific hydrogen bond to a conserved acidic residue in the target affinity and specificity. Our ab initio molecular dynamics simulations reveal that this bond belongs to the SSHB category and accounts for a substantial fraction of the target binding free energy. We also demonstrate that the presence of an extra acidic residue, i.e., aspartic acid at position 32 (D32), found exclusively in mycobacteria, cooperatively enhances the HB strength, ensuring specificity for the mycobacterial target. Consistently, we show that the removal of D32 markedly weakens the affinity, leading to Bq resistance associated with mutations of D32 to nonacidic residues. By designing simple Bq analogs, we then explore the possibility to overcome the resistance and potentially broaden the Bq antimicrobial spectrum by making the SSHB independent of the presence of the extra acidic residue.

Chemical Investigation on the Mechanism and Kinetics of the Atmospheric Degradation Reaction of Trichlorofluoroethene by OH⋠and Its Subsequent Fate in the Presence of O2 /NOx.

The M06-2X/6-311++G(d,p) level of theory was used to examine the degradation of Trichlorofluoroethene (TCFE) initiated by OH⋅ radicals. Additionally, the coupled-cluster single-double with triple perturbative [CCSD(T)] method was employed to refine the single-point energies using the complete basis set extrapolation approach. The results indicated that OH-addition is the dominant pathway. OH⋅ adds to both the C1 and C2 carbons, resulting in the formation of the C(OH)Cl2 -⋅CClF and ⋅CCl2 -C(OH)ClF species. The associated barrier heights were determined to be 1.11 and -0.99 kcal mol-1 , respectively. Furthermore, the energetic and thermodynamic parameters show that pathway 1 exhibits greater exothermicity and exergonicity compared to pathway 2, with differences of 8.11 and 8.21 kcal mol-1 , correspondingly. The primary pathway involves OH addition to the C2 position, with a rate constant of 6.2×10-13 cm3 molecule-1 sec-1 at 298 K. This analysis served to estimate the atmospheric lifetime, along with the photochemical ozone creation potential (POCP) and ozone depletion potential (ODP). It yielded an atmospheric lifetime of 8.49 days, an ODP of 4.8×10-4 , and a POCP value of 2.99, respectively. Radiative forcing efficiencies were also estimated at the M06-2X/6-311++G(d,p) level. Global warming potentials (GWPs) were calculated for 20, 100, and 500 years, resulting in values of 9.61, 2.61, and 0.74, respectively. TCFE is not expected to make a significant contribution to the radiative forcing of climate change. The results obtained from the time-dependent density functional theory (TDDFT) indicated that TCFE and its energized adducts are unable to photolysis under sunlight in the UV and visible spectrum. Secondary reactions involve the [TCFE-OH-O2 ]⋅ peroxy radical, leading subsequently to the [TCFE-OH-O]⋅ alkoxy radical. It was found that the alkoxy radical resulting from the peroxy radical can lead to the formation of phosgene (COCl2 ) and carbonyl chloride fluoride (CClFO), with phosgene being the primary product.

Analysis of Reconstituted Tripartite Complex Supports Avidity-based Recruitment of Hsp70 by Substrate Bound J-domain Protein.

Hsp70 are ubiquitous, versatile molecular chaperones that cyclically interact with substrate protein(s). The initial step requires synergistic interaction of a substrate and a J-domain protein (JDP) cochaperone, via its J-domain, with Hsp70 to stimulate hydrolysis of its bound ATP. This hydrolysis drives conformational changes in Hsp70 that stabilize substrate binding. However, because of the transient nature of substrate and JDP interactions, this key step is not well understood. Here we leverage a well characterized Hsp70 system specialized for iron-sulfur cluster biogenesis, which like many systems, has a JDP that binds substrate on its own. Utilizing an ATPase-deficient Hsp70 variant, we isolated a Hsp70-JDP-substrate tripartite complex. Complex formation and stability depended on residues previously identified as essential for bipartite interactions: JDP-substrate, Hsp70-substrate and J-domain-Hsp70. Computational docking based on the established J-domain-Hsp70(ATP) interaction placed the substrate close to its predicted position in the peptide-binding cleft, with the JDP having the same architecture as when in a bipartite complex with substrate. Together, our results indicate that the structurally rigid JDP-substrate complex recruits Hsp70(ATP) via precise positioning of J-domain and substrate at their respective interaction sites - resulting in functionally high affinity (i.e., avidity). The exceptionally high avidity observed for this specialized system may be unusual because of the rigid architecture of its JDP and the additional JDP-Hsp70 interaction site uncovered in this study. However, functionally important avidity driven by JDP-substrate interactions is likely sufficient to explain synergistic ATPase stimulation and efficient substrate trapping in many Hsp70 systems.

Molecular mechanism and energetics of coupling between substrate binding and product release in the F1-ATPase catalytic cycle.

F1-ATPase is a motor protein that couples the rotation of its rotary [Formula: see text] subunit with ATP synthesis or hydrolysis. Single-molecule experiments indicate that nucleotide binding and release events occur almost simultaneously during the synthesis cycle, allowing the energy gain due to spontaneous binding of ADP to one catalytic [Formula: see text] subunit to be directly harnessed for driving the release of ATP from another rather than being dissipated as heat. Here, we examine the unknown mechanism of this coupling that is critical for an exceptionally high mechanochemical efficiency of F1-ATPase by means of all-atom free-energy simulations. We find that nondissipative and kinetically fast progression of the motor in the synthesis direction requires a concerted conformational change involving the closure of the ADP-binding [Formula: see text] subunit followed by the gradual opening of the ATP-releasing [Formula: see text] subunit over the course of the 30 to 40° rotary substep of the [Formula: see text] subunit. This rotary substep, preceding the ATP-dependent metastable state, allows for the recovery of a large portion of the ADP binding energy in the conformation of ATP-bound [Formula: see text] that gradually adopts the low-affinity conformation, captured also by the recent cryo-EM structure of this elusive state. The release of ATP from this nearly open conformation leads to its further opening, which enables the progression of the motor to the next catalytic metastable state. Our simulations explain this energy conversion mechanism in terms of intersubunit and ligand-protein interactions.

How acidic amino acid residues facilitate DNA target site selection.

Despite the negative charge of the DNA backbone, acidic residues (Asp/Glu) commonly participate in the base readout, with a strong preference for cytosine. In fact, in the solved DNA/protein structures, cytosine is recognized almost exclusively by Asp/Glu through a direct hydrogen bond, while at the same time, adenine, regardless of its amino group, shows no propensity for Asp/Glu. Here, we analyzed the contribution of Asp/Glu to sequence-specific DNA binding using classical and ab initio simulations of selected transcription factors and found that it is governed by a fine balance between the repulsion from backbone phosphates and attractive interactions with cytosine. Specifically, Asp/Glu lower the affinity for noncytosine sites and thus act as negative selectors preventing off-target binding. At cytosine-containing sites, the favorable contribution does not merely rely on the formation of a single H-bond but usually requires the presence of positive potential generated by multiple cytosines, consistently with the observed excess of cytosine in the target sites. Finally, we show that the preference of Asp/Glu for cytosine over adenine is a result of the repulsion from the adenine imidazole ring and a tendency of purine-purine dinucleotides to adopt the BII conformation.

Antibiotic-sterol interactions provide insight into the selectivity of natural aromatic analogues of amphotericin B and their photoisomers.

Aromatic heptaene macrolides (AHMs) belong to the group of polyene macrolide antifungal antibiotics. Members of this group were the first to be used in the treatment of systemic fungal infections. Amphotericin B (AmB), a non-aromatic representative of heptaene macrolides, is of significant clinical importance in the treatment of internal mycoses. It includes the all-trans heptaene chromophore, whereas the native AHMs contain two cis-type (Z) double bonds within the chromophore system. Lately we have proven that it is possible to obtain AHMs' stable derivatives in the form of all-trans (AmB-type) isomers by photochemical isomerization. Our further studies have shown that such alteration leads to the improvement of their selective toxicity in vitro. Computational experiments carried out so far were only an initial contribution in the investigation of the molecular basis of the mechanism of action of AHMs and did not provide explanation to observed differences in biological activity between the native (cis-trans) and isomeric (all-trans) AHMs. Herein, we presented the results of two-dimensional metadynamics studies upon AmB and its aromatic analogues (AHMs), regarding preferable binary antibiotic/sterol complexes orientation, as well as more detailed research on the behaviour of AHMs' alkyl-aromatic side chain in cholesterol- or ergosterol-enriched lipid bilayers.

Guanosine Dianions Hydrated by One to Four Water Molecules.

Intermolecular interactions such as those present in molecule···water complexes may profoundly influence the physicochemical properties of molecules. Here, we carried out an experimental-computational study on doubly deprotonated guanosine monophosphate···water clusters, [dGMP - 2H]2-·nH2O (n = 1-4), using a combination of negative anion photoelectron spectroscopy (NIPES) with molecular dynamics (MD) and quantum chemical (QM) calculations. Successive addition of water molecules to [dGMP - 2H]2- increases the experimental adiabatic detachment (ADE) and vertical detachment energy (VDE) by 0.5-0.1 eV, depending on the cluster size. In order to choose the representative conformations, we combined MD simulations with a clustering procedure to identify low energy geometries for which ADEs and VDEs were computed at the CAM-B3LYP/6-31++G(d,p) level. Our results demonstrate that the assumed approach leads to sound geometries and energetics of the studied microsolvates since the calculated ADEs and VDEs are in pretty good agreement with the experimental characteristics. The evolution of hydrogen bonding with cluster size indicates the possibility of the occurrence of proton transfer for clusters comprising a larger number of water molecules.

Determinants of Directionality and Efficiency of the ATP Synthase Fo Motor at Atomic Resolution.

Fo subcomplex of ATP synthase is a membrane-embedded rotary motor that converts proton motive force into mechanical energy. Despite a rapid increase in the number of high-resolution structures, the mechanism of tight coupling between proton transport and motion of the rotary c-ring remains elusive. Here, using extensive all-atom free energy simulations, we show how the motor's directionality naturally arises from the interplay between intraprotein interactions and energetics of protonation of the c-ring. Notably, our calculations reveal that the strictly conserved arginine in the a-subunit (R176) serves as a jack-of-all-trades: it dictates the direction of rotation, controls the protonation state of the proton-release site, and separates the two proton-access half-channels. Therefore, arginine is necessary to avoid slippage between the proton flux and the mechanical output and guarantees highly efficient energy conversion. We also provide mechanistic explanations for the reported defective mutations of R176, reconciling the structural information on the Fo motor with previous functional and single-molecule data.

Molecular mechanism of proton-coupled ligand translocation by the bacterial efflux pump EmrE.

The current surge in bacterial multi-drug resistance (MDR) is one of the largest challenges to public health, threatening to render ineffective many therapies we rely on for treatment of serious infections. Understanding different factors that contribute to MDR is hence crucial from the global "one health" perspective. In this contribution, we focus on the prototypical broad-selectivity proton-coupled antiporter EmrE, one of the smallest known ligand transporters that confers resistance to aromatic cations in a number of clinically relevant species. As an asymmetric homodimer undergoing an "alternating access" protomer-swap conformational change, it serves as a model for the mechanistic understanding of more complex drug transporters. Here, we present a free energy and solvent accessibility analysis that indicates the presence of two complementary ligand translocation pathways that remain operative in a broad range of conditions. Our simulations show a previously undescribed desolvated apo state and anticorrelated accessibility in the ligand-bound state, explaining on a structural level why EmrE does not disrupt the pH gradient through futile proton transfer. By comparing the behavior of a number of model charged and/or aromatic ligands, we also explain the origin of selectivity of EmrE towards a broad class of aromatic cations. Finally, we explore unbiased pathways of ligand entry and exit to identify correlated structural changes implicated in ligand binding and release, as well as characterize key intermediates of occupancy changes.

Role of cholesterol in substrate recognition by [Formula: see text]-secretase.

[Formula: see text]-Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer's Disease among the most prominent examples. The activity of [Formula: see text]-secretase strictly depends on the membrane cholesterol content, yet the mechanistic role of cholesterol in the substrate binding and cleavage remains unclear. In this work, we used all-atom molecular dynamics simulations to examine the role of cholesterol in the initial binding of a direct precursor of [Formula: see text]-amyloid polypeptides by [Formula: see text]-secretase. We showed that in cholesterol-rich membranes, both the substrate and the enzyme region proximal to the active site induce a local membrane thinning. With the free energy methods we found that in the presence of cholesterol the substrate binds favorably to the identified exosite, while cholesterol depletion completely abolishes the binding. To explain these findings, we directly examined the role of hydrophobic mismatch in the substrate binding to [Formula: see text]-secretase, showing that increased membrane thickness results in higher propensity of the enzyme to bind substrates. Therefore, we propose that cholesterol promotes substrate binding to [Formula: see text]-secretase by increasing the membrane thickness, which leads to the negative hydrophobic mismatch between the membrane and binding partners.

Mechanism of recognition of parallel G-quadruplexes by DEAH/RHAU helicase DHX36 explored by molecular dynamics simulations.

Because of high stability and slow unfolding rates of G-quadruplexes (G4), cells have evolved specialized helicases that disrupt these non-canonical DNA and RNA structures in an ATP-dependent manner. One example is DHX36, a DEAH-box helicase, which participates in gene expression and replication by recognizing and unwinding parallel G4s. Here, we studied the molecular basis for the high affinity and specificity of DHX36 for parallel-type G4s using all-atom molecular dynamics simulations. By computing binding free energies, we found that the two main G4-interacting subdomains of DHX36, DSM and OB, separately exhibit high G4 affinity but they act cooperatively to recognize two distinctive features of parallel G4s: the exposed planar face of a guanine tetrad and the unique backbone conformation of a continuous guanine tract, respectively. Our results also show that DSM-mediated interactions are the main contributor to the binding free energy and rely on making extensive van der Waals contacts between the GXXXG motifs and hydrophobic residues of DSM and a flat guanine plane. Accordingly, the sterically more accessible 5'-G-tetrad allows for more favorable van der Waals and hydrophobic interactions which leads to the preferential binding of DSM to the 5'-side. In contrast to DSM, OB binds to G4 mostly through polar interactions by flexibly adapting to the 5'-terminal guanine tract to form a number of strong hydrogen bonds with the backbone phosphate groups. We also identified a third DHX36/G4 interaction site formed by the flexible loop missing in the crystal structure.

Light-Modulated Sunscreen Mechanism in the Retina of the Human Eye.

The functioning of the human eye in the extreme range of light intensity requires a combination of the high sensitivity of photoreceptors with their photostability. Here, we identify a regulatory mechanism based on dynamic modulation of light absorption by xanthophylls in the retina, realized by reorientation of pigment molecules induced by trans-cis photoisomerization. We explore this photochemically switchable system using chromatographic analysis coupled with microimaging based on fluorescence lifetime and Raman scattering, showing it at work in both isolated human retina and model lipid membranes. The molecular mechanism underlying xanthophyll reorientation is explained in terms of hydrophobic mismatch using molecular dynamics simulations. Overall, we show that xanthophylls in the human retina act as "molecular blinds", opening and closing on a submillisecond timescale to dynamically control the intensity of light reaching the photoreceptors, thus enabling vision at a very low light intensity and protecting the retina from photodegradation when suddenly exposed to strong light.

Two Bacterial Small Heat Shock Proteins, IbpA and IbpB, Form a Functional Heterodimer.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. ß-hairpins formed by strands ß5 and ß7 of IbpA or IbpB crystallin domains associate with the other one's ß-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.

Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA.

An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.

Self-assembly, stability and conductance of amphotericin B channels: bridging the gap between structure and function.

Amphotericin B (AmB), one of the most powerful but also toxic drugs used to treat systemic mycoses, is believed to selectively permeabilize fungal cell membranes to ions in a sterol-dependent manner. Unfortunately, the structure of the biologically active AmB channels has long eluded researchers, obstructing the design of safer alternatives. Here, we investigate the structural and thermodynamic aspects of channel formation, stability, and selective ion conduction. We combine fluorescence lifetime imaging and molecular simulations to trace the process of channel assembly until the formation of stable, roughly octameric double-length channels (DLCs). This stoichiometry is confirmed by matching the predicted channel conductances with the past results of patch-clamp measurements. We then use free energy calculations to explain the effect of sterols on DLC stability and discuss the observed cation selectivity in structural terms, addressing several long-standing controversies in the context of their physiological relevance. Simulations of ion permeation indicate that only solvated ions pass through DLCs, revealing surprising solvation patterns in the channel lumen. We conclude our investigation by inspecting the role of the tail hydroxyl in the assembly of functional channels, pointing at possible origins of the cholesterol-ergosterol selectivity.