Synaptotagmin 7 C2 domains induce membrane curvature stress via electrostatic interactions and the wedge mechanism.

Synaptotagmin 7 (Syt-7) is part of the synaptotagmin protein family that regulates exocytotic lipid membrane fusion. Among the family, Syt-7 stands out by its membrane binding strength and stabilization of long-lived membrane fusion pores. Given that Syt-7 vesicles form long-lived fusion pores, we hypothesize that its interactions with the membrane stabilize the specific curvatures, thicknesses, and lipid compositions that support a metastable fusion pore. Using all-atom molecular dynamics simulations and FRET-based assays of Syt-7's membrane-binding C2 domains (C2A and C2B), we found that Syt-7 C2 domains sequester anionic lipids, are sensitive to cholesterol, thin membranes, and generate lipid membrane curvature by two competing, but related mechanisms. First, Syt-7 forms strong electrostatic contacts with the membrane, generating negative curvature stress. Second, Syt-7's calcium binding loops embed in the membrane surface, acting as a wedge to thin the membrane and induce positive curvature stress. These curvature mechanisms are linked by the protein insertion depth as well as the resulting protein tilt. Simplified quantitative models of the curvature-generating mechanisms link simulation observables to their membrane-reshaping effectiveness.

A conserved electrostatic membrane-binding surface in synaptotagmin-like proteins revealed using molecular phylogenetic analysis and homology modeling.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among the vertebrate synaptotagmin-like protein (Slp) family. Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function.

A Conserved Electrostatic Membrane-Binding Surface in Synaptotagmin-Like Proteins Revealed Using Molecular Phylogenetic Analysis and Homology Modeling.

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among vertebrate synaptotagmin-like proteins (Slps). Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function.

Anti-Leishmania amazonensis Activity, Cytotoxic Features, and Chemical Profile of Allium sativum (Garlic) Essential Oil.

Human tegumentary leishmaniasis (HTL) is a serious tropical disease caused by Leishmania amazonensis. Developing new leishmanicidal agents can help overcome current treatment challenges, such as drug resistance and toxicity. Essential oils are a source of lipophilic substances with diverse therapeutic properties. This study aimed to determine the anti-L. amazonensis activity, cytotoxicity, and chemical profile of Allium sativum essential oil (ASEO). The effect of ASEO on parasite and mammalian cells viability was evaluated using resazurin and MTT assays, respectively. The oil's effect against intracellular amastigotes was also determined. Transmission electron microscopy was used to assess the ultrastructural changes induced by ASEO. In addition, the chemical constituents of ASEO were identified by gas chromatography-mass spectrometry (GC-MS). The cytotoxic potential was evaluated in vitro and in silico. The oil displayed IC50 of 1.76, 3.46, and 3.77 µg/mL against promastigotes, axenic, and intracellular amastigotes, respectively. Photomicrographs of treated parasites showed plasma membrane disruption, increased lipid bodies, and autophagic-like structures. ASEO chemical profiling revealed 1,2,4,6-tetrathiepane (24.84%) and diallyl disulfide (16.75%) as major components. Computational pharmacokinetics and toxicological analysis of ASEO's major components demonstrated good oral bioavailability and better toxicological endpoints than the reference drugs. Altogether, the results suggest that ASEO could be an alternative drug candidate against HTL.

A Survey on the Impact of Intelligent Surfaces in the Terahertz Communication Channel Models.

Terahertz (THz) band will play an important role in enabling sixth generation (6G) envisioned applications. Compared with lower frequency signals, THz waves are severely attenuated by the atmosphere temperature, pressure, and humidity. Thus, designing a THz communication system must take into account how to circumvent or diminish those issues to achieve a sufficient quality of service. Different solutions are being analyzed: intelligent communication environments, ubiquitous artificial intelligence, extensive network automation, and dynamic spectrum access, among others. This survey focuses on the benefits of integrating intelligent surfaces (ISs) and THz communication systems by providing an overview of IS in wireless communications with the scanning of the recent developments, a description of the architecture, and an explanation of the operation. The survey also covers THz channel models, differentiating them based on deterministic and statistical channel modeling. The IS-aided THz channels are elucidated at the end of the survey. Finally, discussions and research directions are given to help enrich the IS field of research and guide the reader through open issues.

Users of cannabis-only are less likely to accept brief interventions than other substance use profiles in a sample of people living with HIV/AIDS.

BACKGROUND AND OBJECTIVES: Research has shown that people living with HIV/AIDS (PLWHA) engage in increased rates of substance use, which has a number of potential negative health outcomes. Increased legalization of cannabis is likely to further increase the availability and use of cannabis in this population. Efforts have been made to integrate screening and intervention resources as part of an individual's routine healthcare visits. Though brief approaches such as Screening and Brief Intervention (SBIRT) have shown promise in addressing alcohol use, results are mixed in addressing cannabis use. The present study investigated how individuals reporting cannabis use responded to an invitation to engage in a brief negotiated intervention (BNI). METHODS: PLWHA participated in a self-administered tablet computer-based version of SBIRT. Patients screened as having at-risk, high-risk, or dependent substance use (N = 331) were eligible to receive the BNI. Of these patients, 101 reported cannabis-only use, with or without alcohol. RESULTS: Binary logistic regressions controlling for Alcohol Use Disorders Identification Test and Drug Abuse Screening Test score and demographics, found that cannabis-only use was significantly related to declining the BNI. DISCUSSION AND CONCLUSIONS: Cannabis-only engagement predicts lower BNI acceptance rates than other substance use profiles; inappropriate screening tools may be one reason for this discrepancy. Implications and directions for future research are discussed. SCIENTIFIC SIGNIFICANCE: Findings are relevant in modifying SBIRT for cannabis use. To our knowledge, this is the first work to evaluate acceptance of brief interventions for cannabis as compared to other substances and brief intervention acceptance in a sample of PLWHA.

Hybrid abutment during prosthetic planning and oral rehabilitation.

INTRODUCTION: The present study aims to describe through a literature review, the characteristics and properties of hybrid abutments, as well as their proper use as a new rehabilitation strategy. EVIDENCE ACQUISITION: A bibliographic search was conducted in the main health databases Pubmed (www.pubmed.gov) and Google Scholar (www.scholar.google.com.br), in which studies published from 2001 to 2020 were collected. Laboratory studies, case reports, systematic and literature reviews were included. Therefore, articles that do not address the characteristics and properties of hybrid abutments were excluded. In addition, studies that did not report the use of hybrid abutments as a new rehabilitation strategy. EVIDENCE SYNTHESIS: According to the inclusion and exclusion criteria, 80 research articles were selected and 20 were excluded, while 25 in vitro, 17 in vivo and 9 in silico studies were reviewed. CONCLUSIONS: The literature demonstrates that hybrid abutments are an excellent alternative in cases of implant-supported rehabilitation, presenting high esthetic results, associated with good soft tissue response, peri implant marginal bone stability and adequate stress distribution during the masticatory loads dissipation.

Challenges to Conducting Contingency Management Treatment for Substance Use Disorders: Practice Recommendations for Clinicians.

A growing empirical literature supports contingency management (CM) as an efficacious treatment for substance use disorders, especially when reinforcers are immediate, frequent, and of sufficient magnitude on escalating schedules. However, in real world-practice, CM is often conducted in ways that are inconsistent with research protocols. One reason for these inconsistencies may be due to pragmatic challenges inherent in conducting CM. In this article, we described an outpatient CM treatment program for drug use disorders and several specific challenges associated with adherence to CM parameters from research protocols. Finally, we propose possible solutions for these challenges and discuss implications for practice.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin.

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

Effect of neutralization and hydrofluoric acid precipitate remotion on the compressive strength of monolithic lithium disilicate crowns.

BACKGROUND: The cleaning protocol for the ceramic surface after acid etching resulted in a decrease in bond strength and flexural strength of a glass ceramic. This study aims to evaluate the effect of different ceramic surface treatments after hydrofluoric acid etching (HF) on the compressive strength of monolithic lithium disilicate crowns. METHODS: Forty (40) human third molars received conventional full coverage preparation. After performing digital impressions of teeth preparations, ceramic blocks were machined using a CAD/CAM system in order to obtain the crowns. The crowns were distributed in 4 groups as ceramic surface treatment (N.=10): (HF) - 4.9% HF for 20s + air-water spray for 30s; (HFN) - HF + neutralizing agent for 5 min (N); (HFU) - HF + ultrasonic bath for 5 min (U); e (HFNU) - HF + N + U. SEM and EDS analysis was performed in each group in order to characterize the ceramic surface and to verify the chemical element distribution after HF cleaning protocols. A silane layer was applied (for 60s), and crowns were then cemented with dual resin cement. A compressive load was applied on the middle of the occlusal crown surface with a crosshead speed of 1 mm/min until fracture. Data were analyzed using ANOVA and Tukey test (α=0.05). RESULTS: Fluoride ions were found in samples of all postetching cleaning protocols. The mean value (Kgf) was: HF =169.92±21.37; HFN =187.34±34.79; HFU =166.63±40.22 and HFNU=175.26±40.22. The ceramic surface treatment after HF etching did not significantly influence (P>0.05) the compressive strength of the tested ceramic crowns. CONCLUSIONS: Surface treatments with neutralizing agent associated with the ultrasonic bath as the pre-cementation protocol was the most efficient protocol in eliminating the precipitate deposited on the porosities created by acid etching.

Evaluation of temperature and osteotomy speed with piezoelectric system.

BACKGROUND: Piezosurgery is an option to realize several clinical and surgical procedures, due to its advantages as precision in osteotomy. This study aims to evaluate the heating and osteotomy speed in bone blocks of ox's shins, to report the best way of its use in the clinical practice. METHODS: A bone blocks had the dimensions as follow: 20 mm length, 10 mm width, and 5 mm wide. It was evaluated 5 different groups: group LM (low speed and medium pressure); group HM (high speed and medium pressure); group HH (high speed and high pressure); group LH (low speed and high pressure); group LL (low speed and low pressure). The heating increasement was measured with a thermal viewer and the osteotomy was timed when the cut depth reached 5 mm and the whole block detached itself. One-way ANOVA and Tukey tests were adopted to analyze the data and the level of significance was set at a P value of 0.05. RESULTS: The pressure and speed of the tip, works directly in the generated temperature during osteotomy. The medium pressure level is the most favorable, because high pressure level caused a high increase in heating over the bone and low pressure presented a very long osteotomy time. CONCLUSIONS: The high speed and medium pressure can be suggested as the most efficient in both standards of time/temperature to realize the osteotomy.

Herbal Medicinal Products from Passiflora for Anxiety: An Unexploited Potential.

Herbal medicines containing Passiflora species have been widely used to treat anxiety since ancient times. The species Passiflora incarnata L. is included in many Pharmacopoeias, and it is the most used species in food, cosmetic, and pharmaceutical industries. However, there are around 600 species of the genus Passiflora and probably other species that can be used safely. Thus, this article was based on a search into the uses of the main species of the genus Passiflora with anxiolytic activity and its main secondary metabolites and some pharmacological studies, patents, and registered products containing Passiflora. Furthermore, the Brazilian Regulatory Health Agency Datavisa, Medicines and Healthcare Products Regulatory Agency of the United Kingdom, and the European Medicines Agency websites were consulted. The results showed that Passiflora species have health benefits but clinical trials are still scarce. The complexity of Passiflora extracts creates challenges for the development of herbal medicines. P. incarnata is the most studied species of the genus and the most used in natural anxiolytic herbal medicine formulations. However, there are hundreds of Passiflora species potentially useful for medicinal and nutraceutical purposes that are still little explored.

β-Carboline-1-propionic acid alkaloid: antileishmanial and cytotoxic effects

ABSTRACT Pentavalent antimonials and amphotericin B remain as the main drugs to treat human leishmaniasis. However, the high toxicity and variable efficacy of treatment have stimulated the search for novel drug candidates. Naturally occurring alkaloids have a long history of antileishmanial activity. Here, we investigate the effects of the β-carboline-1-propionic acid alkaloid isolated from Quassia amara L., Simaroubaceae, against Leishmania amazonensis and Leishmania infatum. The alkaloid was isolated after liquid-liquid fractionation followed by chromatographic purification of the Q. amara methanol extract. The antileishmanial activity was evaluated by the microdilution method, using resazurin as the viability indicator. In addition, annexin and propidium iodide were used to detect parasites undergoing apoptosis. The anti-amastigote activity of the β-carboline-1-propionic acid alkaloid was determined by the infection of RAW 264.7 macrophages. The alkaloid displayed leishmanicidal activity against Leishmania amazonensis and L. infantum promastigotes and intracellular amastigotes with 50% inhibitory concentration ranging from 2.7 ± 0.82 to 9.4 ± 0.5 µg/ml and selectivity indexes >10. Moreover, apoptotic Leishmania amazonensis (19.5%) and L. infantum (40.4%) promastigotes were detected after 5 h incubation with the alkaloid. Finally, the β-carboline-1-propionic acid alkaloid inhibited the production of NO of infected macrophages, suggesting that the intracellular amastigote elimination occurs in a nitrosative stress-independent way. The results shown here suggest that the β-carboline-1-propionic acid alkaloid has potential as an antileishmanial agent.

Membrane-Binding Cooperativity and Coinsertion by C2AB Tandem Domains of Synaptotagmins 1 and 7.

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca2+ to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca2+ concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca2+ titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP2) with greater Ca2+ sensitivity than either of its individual domains and binds to membranes containing PIP2 even in the absence of Ca2+. Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP2-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.

Antimicrobial Potential of Laser Diode in Infected Dentin.

AIM: To evaluate the antibacterial effect of diode laser, associated or not with 2.5% sodium hypochlorite (NaOCl), against Enterococcus faecalis. MATERIALS AND METHODS: Eighty dentin blocks were obtained from single-rooted human teeth and sterilized. Seventy were inoculated with 0.01 mL of fresh bacterial inoculum (within 24 hours of preparation from pure culture) standardized to 1 McFarland turbidity. Contaminated blocks were incubated for 7 days at 37°C in humid conditions. Ten uncontaminated samples were incubated at 37°C during the contamination period to serve as a negative control group, while 10 of the infected specimens served as a positive control group. The dentin blocks were randomly divided into eight experimental groups (n = 10 each) according to the method of decontamination: 2.5% NaOCl alone; 2.5% NaOCl + photodynamic therapy (PDT) with methylene blue/660 nm laser at 18 J for 180 seconds; 2.5% NaOCl + PDT with methylene blue/660 nm laser at 8 J for 80 seconds; methylene blue alone; PDT alone with methylene blue/660 nm laser at 18 J for 180 seconds; PDT alone with methylene blue/660 nm laser at 8 J laser for 80 seconds; positive control group; and negative control group. Microbial growth was evaluated by culture medium turbidity and microbial concentration was analyzed by UV spectrophotometry (adjusted to read at wavelength l = 600 nM). RESULTS: Root canals treated with laser alone at 18 J for 180 seconds had higher bacterial contamination compared with groups in which NaOCl was used, with or without laser irradiation at 18 J for 180 seconds (p < 0.05). CONCLUSION: Photodynamic therapy with a 660 nm diode laser effectively reduced E. faecalis contamination. These findings can guide development of further studies in search of better alternatives for endodontic treatment. CLINICAL RELEVANCE: Chemical and mechanical root canal preparation plays an essential role in reducing microbial burden. However, microorganisms present in areas not mechanically reachable by endodontic instruments. As an alternative to fix this problem, the laser can be applied.

A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling.

Fusion and fission of cellular membranes involve dramatic, protein-mediated changes in membrane curvature. Many of the experimental methods useful for investigating curvature sensing or generation require specialized equipment. We have developed a system based on supported lipid bilayers (SLBs) in which lipid tubules are simple to produce and several types of membrane remodeling events can be readily imaged using widely available instrumentation (e.g., tubule fission and/or membrane budding). Briefly, high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the SLB. After sequentially washing with water and physiological ionic strength buffer solutions, lipid tubules form spontaneously. We find that tubule formation results from solution-dependent spreading of the SLB; washing from water into physiological ionic strength buffer solution leads to expansion of the bilayer and formation of tubules. Conversely, washing from physiological buffer into water results in contraction of the membrane and loss of tubules. We demonstrate the utility of these supported tubulated bilayers, termed "STuBs," with an investigation of Sar1B, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1B to STuBs results in dramatic changes in tubule topology and eventual tubule fission. Overall, STuBs are a simple experimental system, useful for monitoring protein-mediated effects on membrane topology in real time, under physiologically relevant conditions.

The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis.

Synaptotagmin (Syt) proteins comprise a 17-member family, many of which trigger exocytosis in response to calcium. Historically, most studies have focused on the isoform Syt-1, which serves as the primary calcium sensor in synchronous neurotransmitter release. Recently, Syt-7 has become a topic of broad interest because of its extreme calcium sensitivity and diversity of roles in a wide range of cell types. Here, we review the known and emerging roles of Syt-7 in various contexts and stress the importance of its actions. Unique functions of Syt-7 are discussed in light of recent imaging, electrophysiological, and computational studies. Particular emphasis is placed on Syt-7-dependent regulation of synaptic transmission and neuroendocrine cell secretion. Finally, based on biochemical and structural data, we propose a mechanism to link Syt-7's role in membrane fusion with its role in subsequent fusion pore expansion via strong calcium-dependent phospholipid binding.

The synaptotagmin C2B domain calcium-binding loops modulate the rate of fusion pore expansion.

In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca2+-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca2+-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca2+-binding loops of Syt-7 (Syt-1:7C2B123) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B123 and WT Syt-1 are likely to differ in their interactions with Ca2+ and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion.

Lipid-Coated Gold Nanoparticles and FRET Allow Sensitive Monitoring of Liposome Clustering Mediated by the Synaptotagmin-7 C2A Domain.

Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Förster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein-membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca2+ requirements: the threshold Ca2+ concentration for membrane binding is lowest, followed by apposition and aggregation. However, aggregation is highly sensitive to protein concentration and can occur even at submicromolar Syt7 C2A; thus, highly sensitive assays are needed for measuring apposition without complications arising from aggregation. Notably, the localized surface plasmon resonance of the LCAuNP is sensitive to ≤10 nM Syt7 C2A concentrations. Furthermore, when the LCAuNPs were added into a FRET-based liposome apposition assay, the resultant energy transfer increased; possible explanations are discussed. Overall, LCAuNP-based methods allow for highly sensitive detection of protein-induced membrane apposition under conditions that miminize large-scale aggregation.

Is more better? A comparison of tri- and tetrapeptidic catalysts.

From an enzymatic perspective, there is a general notion that the bigger and more complex a catalytically active peptide is the more enzyme-like and the better it should become. But is this really true? We have tackled this question firstly by screening split-and-mix-libraries of tri- and tetrapeptides for members that catalyze aldol reactions. Then, the catalytic performance of all possible diastereoisomers of related tri- and tetrapeptidic catalysts of the type H-Pro-Pro-Glu/Asp-NH2 and H-Pro-Pro-Glu/Asp-Pro-NH2 in aldol and conjugate addition reactions was compared.