Applications of the Newly Developed Force-Field Parameters Uncover a Dynamic Nature of Ω-Loop C in the Lys-Ligated Alkaline Form of Cytochrome c.

Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c-type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso-1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.

Lipid-mediated hydrophobic gating in the BK potassium channel.

The large-conductance, calcium-activated potassium (BK) channel lacks the typical intracellular bundle-crossing gate present in most ion channels of the 6TM family. This observation, initially inferred from Ca$^{2+}$-free-pore accessibility experiments and recently corroborated by a CryoEM structure of the non-conductive state, raises a puzzling question: how can gating occur in absence of steric hindrance? To answer this question, we carried out molecular simulations and accurate free energy calculations to obtain a microscopic picture of the sequence of events that, starting from a Ca$^{2+}$-free state leads to ion conduction upon Ca$^{2+}$ binding. Our results highlight an unexpected role for annular lipids, which turn out to be an integral part of the gating machinery. Due to the presence of fenestrations, the "closed" Ca$^{2+}$-free pore can be occupied by the methyl groups from the lipid alkyl chains. This dynamic occupancy triggers and stabilizes the nucleation of a vapor bubble into the inner pore cavity, thus hindering ion conduction. By contrast, Ca$^{2+}$ binding results into a displacement of these lipids outside the inner cavity, lowering the hydrophobicity of this region and thus allowing for pore hydration and conduction. This lipid-mediated hydrophobic gating rationalizes several seemingly problematic experimental observations, including the state-dependent pore accessibility of blockers.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore region.

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.

Dual mechanisms contribute to enhanced voltage dependence of an electric fish potassium channel.

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel's voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

Unplugging lateral fenestrations of NALCN reveals a hidden drug binding site within the pore module.

The sodium (Na + ) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (Na V s and Ca V s, respectively). Unlike Na V s and Ca V s, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145 and Y1436) that block these openings. Structural data suggest that oc-cluded lateral fenestrations underlie the pharmacological resistance of NALCN to lipophilic compounds, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned resi-dues with alanine (AAAA) and compared the effects of Na V , Ca V and NALCN block-ers on both wild-type (WT) and AAAA channels. Most compounds behaved in a simi-lar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cav-ity through distinct fenestrations, implying structural specificity to their modes of ac-cess. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug bind-ing site(s) within the pore region. Intrigued by the activity of 2-APB and its ana-logues, we tested additional compounds containing the diphenylmethane/amine moiety on WT channels. We identified compounds from existing clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the resistance of NALCN to pharmacological targeting, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties. Significance statement: The sodium leak channel (NALCN) is essential for survival: mutations cause life-threatening developmental disorders in humans. However, no treatment is currently available due to the resistance of NALCN to pharmacological targeting. One likely reason is that the lateral fenestrations, a common route for clinically used drugs to enter and block related ion channels, are occluded in NALCN. Using a combination of computational and functional approaches, we unplugged the fenestrations of NALCN which led us to the first molecularly defined drug binding site within the pore region. Besides that, we also identified additional NALCN modulators from existing clinically used therapeutics, thus expanding the pharmacological toolbox for this leak channel.

Carotid intima-media thickness in surgically or conservatively managed patients with primary hyperparathyroidism.

CONTEXT: Current evidence of cardiovascular (CV) risk in primary hyperparathyroidism (PHPT) is still inconsistent. OBJECTIVE: To prospectively investigate changes of early atherosclerosis in patients with PHPT undergoing parathyroidectomy (PTx) or conservative management, according to Consensus criteria. METHODS: Biochemical parameters of PHPT, CV risk factors (systolic and diastolic blood pressure-BP-, total-, HDL- and LDL-cholesterol, triglyceride, glycosilated hemoglobin, and HOMA-IR), and carotid intima-media thickness (IMT) and plaque were assessed in 52 consecutive postmenopausal PHPT patients both at baseline and ≥24 months after surgery (PTx: n = 22) or conservative management (no-PTx: n = 30). RESULTS: At baseline, PTx and no-PTx showed comparable age, BMI, renal function, 25(OH)D levels, and did not differ for CV risk factors, IMT and plaques, nor for the prevalence of smoking, diabetes mellitus, antihypertensive or statin therapy, while differing for all parameters characterizing PHPT. Follow-up length in PTx was longer (p = 0.004) than in no-PTx. Parameters characterizing PHPT significantly improved ≥24 months after surgery, whereas in no-PTx serum phosphate slightly decreased and PTH increased. Systolic and diastolic BP increased at follow-up in both groups, while other CV risk factors did not significantly vary. In PTx IMT did not significantly vary after surgery (0.85 ± 0.14 to 0.89 ± 0.22 mm, p = 0.366), whereas it significantly increased in no-PHPT (0.80 ± 0.18 to 0.93 ± 0.23 mm, p = 0.008), even adjusting for BP values. Plaque prevalence and incidence did not significantly differ in the two groups. CONCLUSION: Our results suggest that in postmenopausal PHPT patients subclinical atherosclerosis could be halted by PTx, whereas it worsens over time in not operated patients with milder disease.

Cation Modifies Interfacial Water Structures on Platinum during Alkaline Hydrogen Electrocatalysis.

The molecular details of an electrocatalytic interface play an essential role in the production of sustainable fuels and value-added chemicals. Many electrochemical reactions exhibit strong cation-dependent activities, but how cations affect reaction kinetics is still elusive. We report the effect of cations (K+, Li+, and Ba2+) on the interfacial water structure using second-harmonic generation (SHG) and classical molecular dynamics (MD) simulation. The second- (χH2O(2)) and third-order (χH2O(3)) optical susceptibilities of water on Pt are smaller in the presence of Ba2+ compared to those of K+, suggesting that cations can affect the interfacial water orientation. MD simulation reproduces experimental SHG observations and further shows that the competition between cation hydration and interfacial water alignment governs the net water orientation. The impact of cations on interfacial water supports a cation hydration-mediated mechanism for hydrogen electrocatalysis; i.e., the reaction occurs via water dissociation followed by cation-assisted hydroxide/water exchange on Pt. Our study highlights the role of interfacial water in electrocatalysis and how innocent additives (such as cations) can affect the local electrochemical environment.

Calcium-phosphate homeostasis and insulin resistance in men.

BACKGROUND AND AIMS: Data on P homeostasis in insulin resistance (IR) are still conflicting. We investigated calcium-phosphate homeostasis parameters in men with/without IR. METHODS AND RESULTS: 177 volunteers (aged 61.62 ± 12.11), whose body mass index (BMI) was 29.97 ± 6.35, were studied. On fasting blood and spot urine samples, we measured serum creatinine, sodium (sNa), potassium (sK), chloride (sCl), calcium (sCa), phosphate (sP), alkaline phosphatase total activity (ALP), glucose, insulin, parathyroid hormone (PTH), 25-hydroxy-vitamin D [25(OH)D], and urinary electrolytes corrected for creatinine (uNa/Cr, uK/Cr, uCl/Cr, uCa/Cr, and uP/Cr). Through the QUICKI index, we separated subjects with (IR+, n = 68) or without (IR-, n = 109) IR, and their parameters were compared. Associations were assessed by age-adjusted partial correlation, whose coefficients were compared by Fisher's transform. IR + had higher sP (3.54 ± 0.65 vs. 3.35 ± 0.47, p = 0.044) and lower uCa/Cr levels (0.073 ± 0.056 vs. 0.095 ± 0.072, p = 0.047) than IR-. BMI correlated with sP (r = 0.21, p < 0.05) and PTH (r = 0.29, p < 0.01). QUICKI negatively correlated with sCa (r = -0.22, p < 0.05) and positively with uCa/Cr (r = 0.21, p < 0.05), in turn correlating with uNa/Cr (r = 0.45, p < 0.001). In both groups, uCa/Cr correlated with eGFR and uNa/Cr (p < 0.05 to p < 0.001). In IR + only, sP correlated with BMI, PTH with insulin, and uP/Cr (p < 0.05 for all). IR+ and IR-coefficients differed (p < 0.05 to p < 0.001) for the correlation of sP with BMI and of PTH with insulin and uP/Cr. CONCLUSION: The higher sP and lower uCa/Cr levels found in men with IR + suggest that IR could modulate calcium-phosphate homeostasis, likely by affecting their renal handling.

The DTX Protein Family: An Emerging Set of E3 Ubiquitin Ligases in Cancer.

Until recently, Deltex (DTX) proteins have been considered putative E3 ligases, based on the presence of an E3 RING domain in their protein coding sequence. The human DTX family includes DTX1, DTX2, DTX3, DTX3L and DTX4. Despite the fact that our knowledge of this class of E3-ubiquitin ligases is still at an early stage, our understanding of their role in oncogenesis is beginning to unfold. In fact, recently published studies allow us to define specific biological scenarios and further consolidate evidence-based working hypotheses. According to the current evidence, all DTX family members are involved in the regulation of Notch signaling, suggesting a phylogenetically conserved role in the regulation of this pathway. Indeed, additional evidence reveals a wider involvement of these proteins in other signaling complexes and cancer-promoting mechanisms beyond NOTCH signaling. DTX3, in particular, had been known to express two isoform variants (DTX3a and DTX3b). The recent identification and cloning of a third isoform variant in cancer (DTX3c), and its specific involvement in EphB4 degradation in cancer cells, sheds further light on this group of proteins and their specific role in cancer. Herein, we review the cumulative knowledge of this family of E3 Ubiquitin ligases with a specific focus on the potential oncogenic role of DTX isoforms in light of the rapidly expanding findings regarding this protein family's cellular targets and regulated signaling pathways. Furthermore, using a comparative and bioinformatic approach, we here disclose a new putative motif of a member of this family which may help in understanding the biological and contextual differences between the members of these proteins.

Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma.

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

Exploration of the Nucleation Pathway for Supramolecular Fibers.

The pathway for supramolecular fiber formation is coupled with the underlying order of the self-assembling molecules. Here, we report on atomistic molecular dynamics simulations to characterize the initial stages of the self-assembly of a model drug amphiphile in an aqueous solution. We perform two-dimensional metadynamics calculations to characterize the assembly space of this model drug amphiphile─Tubustecan, TT1. TT1 is composed of the hydrophobic anticancer drug, Camptothecin (CPT), conjugated to a hydrophilic polyethylene glycol (PEG) chain. We find that the aromatic stacking of CPT drives the formation of a higher-density liquid droplet. This droplet elongates and can form a higher-ordered supramolecular assembly upon reorganizing and forming an interface and additional aromatic stacking of the drugs. We show that novel reaction coordinates tailored to this class of molecules are essential in capturing the underlying degree of molecular order upon assembly. This approach can be refined and extended to characterize the supramolecular assembly pathway of other molecules containing aromatic compounds.

Molecular Engineering of pH-Responsive Anchoring Systems onto Poly(ethylene glycol) Corona.

An adaptive surface that can sense and respond to environmental stimuli is integral to smart functional materials. Here, we report pH-responsive anchoring systems onto the poly(ethylene glycol) (PEG) corona of polymer vesicles. The hydrophobic anchor, pyrene, is reversibly inserted into the PEG corona through the reversible protonation of its covalently linked pH-sensing group. Depending on the pKa of the sensor, the pH-responsive region is engineered from acidic to neutral and basic conditions. The switchable electrostatic repulsion between the sensors contributes to the responsive anchoring behavior. Our findings provide a new responsive binding chemistry for the creation of smart nanomedicine and a nanoreactor.

Detecting Liquid-Liquid Phase Separations Using Molecular Dynamics Simulations and Spectral Clustering.

A stringent test of the accuracy of empirical force fields is reproducing the phase diagram of bulk phases and mixtures. Exploring the phase diagram of mixtures requires the detection of phase boundaries and critical points. In contrast to most solid-liquid transitions, in which a global order parameter (average density) can be used to discriminate between two phases, some demixing transitions entail relatively subtle changes in the local environment of each molecule. In such cases, finite sampling errors and finite-size effects make the identification of trends in local order parameters extremely challenging. Here we analyze one such example, namely a methanol/hexane mixture, and compute several local and global structural properties. We simulate the system at various temperatures and study the structural changes associated with demixing. We show that despite a seemingly continuous transformation between mixed and demixed states, the topological properties of the H-bond network change abruptly as the system crosses the demixing line. In particular, by using spectral clustering, we show that the distribution of cluster sizes develops a fat tail (as expected from percolation theory) in the vicinity of the critical point. We illustrate a simple criterion to identify this behavior, which results from the emergence of large system-spanning clusters from a collection of aggregates. We further tested the spectral clustering analysis on a Lennard-Jones system as a standard example of a system with no H-bonds, and also, in this case, we were able to detect the demixing transition.

Vitamin D Increases Irisin Serum Levels and the Expression of Its Precursor in Skeletal Muscle.

Irisin is a myokine synthesized by skeletal muscle, which performs key actions on whole-body metabolism. Previous studies have hypothesized a relationship between irisin and vitamin D, but the pathway has not been thoroughly investigated. The purpose of the study was to evaluate whether vitamin D supplementation affected irisin serum levels in a cohort of 19 postmenopausal women with primary hyperparathyroidism (PHPT) treated with cholecalciferol for six months. In parallel, to understand the possible link between vitamin D and irisin, we analyzed the expression of the irisin precursor, Fndc5, in the C2C12 myoblast cell line treated with a biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Our results demonstrate that vitamin D supplementation resulted in a significant increase in irisin serum levels (p = 0.031) in PHPT patients. In vitro, we show that vitamin D treatment on myoblasts enhanced Fndc5 mRNA after 48 h (p = 0.013), while it increased mRNAs of sirtuin 1 (Sirt1) (p = 0.041) and peroxisome proliferator-activated receptor γ coactivator 1α (Pgc1α) (p = 0.017) over a shorter time course. Overall, our data suggest that vitamin-D-induced modulation of Fndc5/irisin occurs through up-regulation of Sirt1, which together with Pgc1α, is an important regulator of numerous metabolic processes in skeletal muscle.

Nonequilibrium Thermodynamics of DNA Nanopore Unzipping.

Using theory and simulations, we carried out a first systematic characterization of DNA unzipping via nanopore translocation. Starting from partially unzipped states, we found three dynamical regimes depending on the applied force f: (i) heterogeneous DNA retraction and rezipping (f<17 pN), (ii) normal (17 pN60 pN) drift-diffusive behavior. We show that the normal drift-diffusion regime can be effectively modeled as a one-dimensional stochastic process in a tilted periodic potential. We use the theory of stochastic processes to recover the potential from nonequilibrium unzipping trajectories and show that it corresponds to the free-energy landscape for single-base-pair unzipping. Applying this general approach to other single-molecule systems with periodic potentials ought to yield detailed free-energy landscapes from out-of-equilibrium trajectories.

Adaptive insertion of a hydrophobic anchor into a poly(ethylene glycol) host for programmable surface functionalization.

Covalent and non-covalent molecular binding are two strategies to tailor surface properties and functions. However, the lack of responsiveness and requirement for specific binding groups makes spatiotemporal control challenging. Here, we report the adaptive insertion of a hydrophobic anchor into a poly(ethylene glycol) (PEG) host as a non-covalent binding strategy for surface functionalization. By using polycyclic aromatic hydrocarbons as the hydrophobic anchor, hydrophilic charged and non-charged functional modules were spontaneously loaded onto PEG corona in 2 min without the assistance of any catalysts and binding groups. The thermodynamically favourable insertion of the hydrophobic anchor can be reversed by pulling the functional module, enabling programmable surface functionalization. We anticipate that the adaptive molecular recognition between the hydrophobic anchor and the PEG host will challenge the hydrophilic understanding of PEG and enhance the progress in nanomedicine, advanced materials and nanotechnology.

Validation of the clinical consensus recommendations on the management of fracture risk in postmenopausal women with type 2 diabetes.

BACKGROUND AND AIMS: Bone fragility is recognized as a complication of type 2 diabetes (T2D). However, the fracture risk in T2D is underestimated using the classical assessment tools. An expert panel suggested the diagnostic approaches for the detection of T2D patients worthy of bone-active treatment. The aim of the study was to apply these algorithms to a cohort of T2D women to validate them in clinical practice. METHODS AND RESULTS: The presence of T2D-specific fracture risk factors (T2D ≥ 10 years, ≥1 T2D complications, insulin or thiazolidinedione use, poor glycaemic control) was assessed at baseline in 107 postmenopausal T2D women. In all patients at baseline and in 34 patients after a median follow-up of 60.2 months we retrospectively evaluated bone mineral density and clinical and morphometric vertebral fractures. No patient was treated with bone-active drug. Following the protocols, 34 (31.8%) and 73 (68.2%) patients would have been pharmacologically and conservatively treated, respectively. Among 49 patients without both clinical fractures and major T2D-related risk factors, who would have been, therefore, conservatively followed-up without vertebral fracture assessment, only one showed a prevalent vertebral fracture (sensitivity 90%, negative predictive value 98%). The two patients who experienced an incident fracture would have been pharmacologically treated at baseline. CONCLUSIONS: The clinical consensus recommendations showed a very good sensitivity in identifying T2D postmenopausal women at high fracture risk. Among those with treatment indication as many as 13% of patients experienced an incident fracture, and, conversely, among those without treatment indication no incident fractures were observed.

Computational and functional studies of the PI(4,5)P2 binding site of the TRPM3 ion channel reveal interactions with other regulators.

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel expressed in peripheral sensory neurons and the central nervous system. TRPM3 activity depends on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but the molecular mechanism of activation by PI(4,5)P2 is not known. As no experimental structure of TRPM3 is available, we built a homology model of the channel in complex with PI(4,5)P2via molecular modeling. We identified putative contact residues for PI(4,5)P2 in the pre-S1 segment, the S4-S5 linker, and the proximal C-terminal TRP domain. Mutating these residues increased sensitivity to inhibition of TRPM3 by decreasing PI(4,5)P2 levels. Changes in ligand-binding affinities via molecular mechanics/generalized Born surface area (MM/GBSA) showed reduced PI(4,5)P2 affinity for the mutants. Mutating PI(4,5)P2-interacting residues also reduced sensitivity for activation by the endogenous ligand pregnenolone sulfate, pointing to an allosteric interaction between PI(4,5)P2 and pregnenolone sulfate. Similarly, mutating residues in the PI(4,5)P2 binding site in TRPM8 resulted in increased sensitivity to PI(4,5)P2 depletion and reduced sensitivity to menthol. Mutations of most PI(4,5)P2-interacting residues in TRPM3 also increased sensitivity to inhibition by Gßγ, indicating allosteric interaction between Gßγ and PI(4,5)P2 regulation. Disease-associated gain-of-function TRPM3 mutations on the other hand resulted in no change of PI(4,5)P2 sensitivity, indicating that mutations did not increase channel activity via increasing PI(4,5)P2 interactions. Our data provide insight into the mechanism of regulation of TRPM3 by PI(4,5)P2, its relationship to endogenous activators and inhibitors, as well as identify similarities and differences between PI(4,5)P2 regulation of TRPM3 and TRPM8.

Molecular states and spin crossover of hemin studied by DNA origami enabled single-molecule surface-enhanced Raman scattering.

The study of biologically relevant molecules and their interaction with external stimuli on a single molecular scale is of high importance due to the availability of distributed rather than averaged information. Surface enhanced Raman scattering (SERS) provides direct chemical information, but is rather challenging on the single molecule (SM) level, where it is often assumed to require a direct contact of analyte molecules with the metal surface. Here, we detect and investigate the molecular states of single hemin by SM-SERS. A DNA aptamer based G-quadruplex mediated recognition of hemin directs its placement in the SERS hot-spot of a DNA Origami Nanofork Antenna (DONA). The configuration of the DONA structure allows the molecule to be trapped at the plasmonic hot-spot preferentially in no-contact configuration with the metal surface. Owing to high field enhancement at the plasmonic hot spot, the detection of a single folded G-quadruplex becomes possible. For the first time, we present a systematic study by SM-SERS where most hemin molecule adopt a high spin and oxidation state (III) that showed state crossover to low spin upon strong-field-ligand binding. The present study therefore, provides a platform for studying biologically relevant molecules and their properties at SM sensitivity along with demonstrating a conceptual advancement towards successful monitoring of single molecular chemical interaction using DNA aptamers.

Superhydrophilicity of α-alumina surfaces results from tight binding of interfacial waters to specific aluminols.

HYPOTHESIS: Understanding the microscopic driving force of water wetting is challenging and important for design of materials. The relations between structure, dynamics and hydrogen bonds of interfacial water can be investigated using molecular dynamics simulations. EXPERIMENTS AND SIMULATIONS: Contact angles at the alumina (0001) and (112‾0) surfaces are studied using both classical molecular dynamics simulations and experiments. To test the superhydrophilicity, the free energy cost of removing waters near the interfaces are calculated using the density fluctuations method. The strength of hydrogen bonds is determined by their lifetime and geometry. FINDINGS: Both surfaces are superhydrophilic and the (0001) surface is more hydrophilic. Interactions between surfaces and interfacial waters promote a templating effect whereby the latter are aligned in a pattern that follows the underlying lattice of the surfaces. Translational and rotational dynamics of interfacial water molecules are slower than in bulk water. Hydrogen bonds between water and both surfaces are asymmetric, water-to-aluminol ones are stronger than aluminol-to-water ones. Molecular dynamics simulations eliminate the impacts of surface contamination when measuring contact angles and the results reveal the microscopic origin of the macroscopic superhydrophilicity of alumina surfaces: strong water-to-aluminol hydrogen bonds.