Complete Solubilization Research Articles

Statins Solubilized in Fish Oil: Versatile and Simple Combinations by Innovative Formulations

Dyslipidemia or hyperlipidemia comprised mainly hypertriglyceridemia and/or hypercholesterolemia; it is now well established as their control is considered to have an impact on public health. Therefore, in an overcrowded field to find appropriate answers to lower blood levels of cholesterol and triglycerides, here we report findings of formulation studies, aimed to combine statins and n-3 PUFA, overcoming drawbacks related to solubility and stability. A new formulation has been made creating a solvent system that allows for a complete solubilization of statin drugs such as atorvastatin, rosuvastatin, and pitavastatin. With simvastatin, a preliminary dehydration with molecular sieves was also needed for stability purpose. These findings are of great importance and could be applied widely in all cases where a co-administration of the two active therapeutic ingredients are routinely required.

Influence of lipid bilayer properties on nanodisc formation mediated by styrene/maleic acid copolymers.

Copolymers of styrene and maleic acid (SMA) have gained great attention as alternatives to conventional detergents, as they offer decisive advantages for studying membrane proteins and lipids in vitro. These polymers self-insert into artificial and biological membranes and, at sufficiently high concentrations, solubilise them into disc-shaped nanostructures containing a lipid bilayer core surrounded by a polymer belt. We have used (31)P nuclear magnetic resonance spectroscopy and dynamic light scattering to systematically study the solubilisation of vesicles composed of saturated or unsaturated phospholipids by an SMA copolymer with a 3 : 1 styrene/maleic acid molar ratio at different temperatures. Solubilisation was thermodynamically rationalised in terms of a three-stage model that treats various lipid/polymer aggregates as pseudophases. The solubilising capacity of SMA(3 : 1) towards a saturated lipid is higher in the gel than in the liquid-crystalline state of the membrane even though solubilisation is slower. Although the solubilisation of mixed fluid membranes is non-selective, the presence of a non-bilayer phospholipid lowers the threshold at which the membrane becomes saturated with SMA(3 : 1) but raises the polymer concentration required for complete solubilisation. Both of these trends can be explained by considering the vesicle-to-nanodisc transfer free energies of the lipid and the polymer. On the basis of the phase diagrams thus obtained, re-association of polymer-solubilised lipids with vesicles is possible under mild conditions, which has implications for the reconstitution of proteins and lipids from nanodiscs into vesicular membranes. Finally, the phase diagrams provide evidence for the absence of free SMA(3 : 1) in vesicular lipid suspensions.

Production of Cloudifier Products from Lemon, Orange, Melon, Persimmon Fruit and its Automation

Objective: An increase of cloud stability and cloud level of cloudifier juices and concentrates of citrus, melon and persimmon fruit considerably increased their sensory characteristics and beneficial health properties. The aim is to develop the cloudifier juice technology using the continuous process of flash vacuum expansion in the industry of cloudifier juice. Materials and methods: A systematic search of the database literature up to 2014 for studies on the cloudifier juice production was conducted according to a methodology developed a priori. The property and cloud stability of cloudifier juices and concentrates from citrus, melon and persimmon fruit was determined using standardized methods. Results: Evidence from the selected studies were used to develop the industry production of cloudifier juices and concentrates from citrus, melon, persimmon fruits The cloudifier juice and concentrate from citrus, melon, persimmon fruits were obtained using the cavitation, enzyme treatment and flash vacuum expansion. The technology relates to the method that prevents the gel formation, intense browning and re-increase of astringency. The few important basic conditions, such as the enzyme treatment at low temperature, 2.8-4.4 mS electrical conductivity and the sudden expansion treatment during 30 min, formed the pectin oligomer with 60% degree of esterification, micelle (1.0-1.3 μm) network. The application of proposed procedure considerably increased the turbidity to 5000-14940 FNU in 8.0-11.2 °Brix reconstituted juice. The thermal treatment of cloudifier samples at 85-95°C not accelerated the sedimentation process. Cloudifier industry concentrates after cooled were stable for longer too. From an engineering point of view, the micelle size to complete solubilization is a critical process indicator and it determined the electrical conductivity. The relationship between turbidity and conductivity in cloudifier juices from citrus, melon, persimmon fruit was offered for control and automation in processing. Conclusions: This technology which is able to modify the pectin chains and form the micelle network was used in the industry production during a large period of time. During these period 60 lots of industrial cloudifier lemon and orange concentrates were analyzed. The cloudifier fruit juice and concentrate from citrus, melon, persimmon fruit has much benefits potential over their fruit juices and concentrates with a higher pulp, such as: • more natural colour • the turbidity more than 56870 FNU in the 8.0-11.2 °Brix reconstituted juice with a low content of the pulp (0-0.4%; 0.04-1.0% in industry products) • longer shelf life In summary of these study findings, it was found that the content of modified pectin was high and this level satisfied the inhibition of cancer cell proliferation. Further studies are required to quantitatively determine the effect of particle size distribution and cloud loss of the cloudifier juice.

Autohydrolysis pretreatment of Arundo donax: a comparison between microwave-assisted batch and fast heating rate flow-through reaction systems.

BackgroundAutohydrolysis of lignocellulosic biomass in liquid hot water has been widely studied owing to its high efficiency and relatively low cost. In the perspective of industrial applications, continuous or semi-continuous processes are more interesting than batch systems. Moreover, microwave heating of pretreatment systems has been proposed to intensify the kinetics of the process. In this study, the autohydrolysis of Arundo donax was performed in pure liquid hot water using a microwave-heated batch reactor and a semi-continuous flow-through reaction system with fast heating rate at the same operating conditions with the aim of performing a systematic comparison between the two different experimental apparatuses.ResultsThe effect of process temperature and time, biomass to water mass to volume ratio and water flow rate on the concentration and yield of hydrolysis products was investigated. The flow-through set-up allowed us to reach biomass solubilization up to 44.5 wt% on dry basis, while the batch system stopped at 34.5 wt% suggesting that the mass transfer could be the rate-determining step in the solubilization of the constituting biopolymers. For example, in the flow-through layout, using a flow rate of 3.5 mL/min at 200 °C with 20 min of processing time, quantitative recovery of hemicellulose was obtained with limited formation of degradation products. Interestingly, higher cellulose/hemicellulose extraction ratios were found using the microwave-assisted batch reactor. FTIR analyses of the solid residues recovered after the pretreatment offered independent information on the fractions of liquefied biopolymers complementary to those derived from HPLC and UV–Vis spectroscopy.ConclusionsCollected experimental results indicated that the flow-through system can be adopted to obtain complete solubilization of the hemicellulose fraction of Arundo donax addressing the product distribution in soluble compounds towards fermentable sugars with limited formation of sugar degradation products and with limited penalty in terms of dilution of the hydrolysate solution. It was also found that microwaves can promote cellulose depolymerization and solubilization, thus allowing a more comprehensive utilization of the biomass and that infrared spectroscopy can be a useful technique to estimate the effect of the pretreatment.

Gel-free mass spectrometry analysis of Drosophila melanogaster heads.

Membrane proteins play key roles in several fundamental biological processes such as cell signalling, energy metabolism and transport. Despite the significance, these still remain an under-represented group in proteomics datasets. Herein, a bottom-up approach to analyse an enriched membrane fraction from Drosophila melanogaster heads using multidimensional liquid chromatography (LC) coupled with tandem-mass spectrometry (MS/MS) that relies on complete solubilisation and digestion of proteins, is reported. An enriched membrane fraction was prepared using equilibrium density centrifugation on a discontinuous sucrose gradient, followed by solubilisation using the filter-aided sample preparation (FASP), tryptic and sequential chymotryptic digestion of proteins. Peptides were separated by reversed-phase (RP) LC at high pH in the first dimension and acidic RP-LC in the second dimension coupled directly to an Orbitrap Velos Pro mass spectrometer. A total number of 4812 proteins from 114 865 redundant and 38 179 distinct peptides corresponding to 4559 genes were identified in the enriched membrane fraction from fly heads. These included brain receptors, transporters and channels that are most important elements as drug targets or are linked to disease. Data are available via ProteomeXchange with identifier PXD001712 (http://proteomecentral.proteomexchange.org/dataset/PXD001712).

Development of a wet digestion method for paints for the determination of metals and metalloids using inductively coupled plasma optical emission spectrometry

Paints, a complex matrix, have a variable composition that is dependent on the application. In this work, a new wet digestion procedure for the determination of Al, As, Ba, Cd, Cr, Cu, Mn, Ni, Pb, Sn, Sr, Ti and Zn in paint samples using inductively coupled plasma optical emission spectrometry (ICP OES) has been developed. An experimental design approach was employed to determine the optimal conditions for achieving complete solubilization and/or decomposition in the sample preparation method. An efficient sample preparation was developed that consisted of a pre-digestion step at 40°C for 20min using 1mL of HNO3 to eliminate organic solvents followed by digestion at 120°C for 3h using 5mL of HCl and 1mL of HF in a block digestion. The proposed procedure promotes the complete solubilization of different bases of paints at low temperature and atmospheric pressure. The accuracy was determined by addition/recovery tests and comparing the results with those obtained using the ASTM D335-85a standard sample preparation method. The limits of quantification were 1.78, 0.11, 0.006, 0.006, 0.01, 0.04, 0.006, 0.006, 0.02, 0.07, 0.30, 1.30 and 0.30mgkg−1 for Al, As, Ba, Cd, Cr, Cu,Mn, Ni, Pb, Sn, Sr, Ti and Zn, respectively. The proposed method was applied for the analysis of inorganics via the ICP OES of paints with different colors and bases used to cover wall surfaces.

Biochemical characterization of GM1 micelles-Amphotericin B interaction.

In this work a thorough characterization of the GM1 micelle-Amphotericin B (AmB) interaction was performed. The micelle formation as well as the drug loading occurs spontaneously, although influenced by the physicochemical conditions, pH and temperature. The chromatographic profile of GM1-AmB complexes at different molar ratios shows the existence of two populations. The differential absorbance of GM1, monomeric and aggregate AmB, allowed us to discriminate the presence of all of them in both fractions. Thus, we noted that at higher proportion of AmB in the complex, increases the larger population which is composed mainly of aggregated AmB. The physical behavior of these micelles shows that both GM1- AmB complexes were stable in solution for at least 30 days. However upon freeze-thawing or lyophilization-solubilization cycles, only the smallest population, enriched in monomeric AmB, showed a complete solubilization. In vitro, GM1-AmB micelles were significantly less toxic on cultured cells than other commercial micellar formulations as Fungizone, but had a similar behavior to liposomal formulations as Ambisome. Regarding the antifungal activity of the new formulation, it was very similar to that of other formulations. The characterization of these GM1-AmB complexes is discussed as a potential new formulation able to improve the antifungal therapeutic efficiency of AmB.

Multicomponent mixtures for cryoprotection and ligand solubilization

Mixed cryoprotectants have been developed for the solubilization of ligands for crystallization of protein–ligand complexes and for crystal soaking. Low affinity lead compounds with poor solubility are problematic for structural studies. Complete ligand solubilization is required for co-crystallization and crystal soaking experiments to obtain interpretable electron density maps for the ligand. Mixed cryo-preserving compounds are needed prior to X-ray data collection to reduce radiation damage at synchrotron sources. Here we present dual-use mixes that act as cryoprotectants and also promote the aqueous solubility of hydrophobic ligands. Unlike glycerol that increases protein solubility and can cause crystal melting the mixed solutions of cryo-preserving compounds that include precipitants and solubilizers, allow for worry-free crystal preservation while simultaneously solubilizing relatively hydrophobic ligands, typical of ligands obtained in high-throughput screening. The effectiveness of these mixture has been confirmed on a human transthyretin crystals both during crystallization and in flash freezing of crystals.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC–MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.

Dissolution of NaCl crystals in artificial saliva and water by video-microscopy

Abstract Excess sodium as salt (NaCl) added to foods may be harmful to humans. Most salt intake comes from processed foods, therefore salt reduction strategies should be developed by the food industry. One strategy seeks to optimize the delivery of salt ions inside the mouth. To accomplish this purpose, the dissolution characteristics of salt crystals in saliva must be understood. However, relatively scarce information is available on this topic. This study compares the dissolution of five commercial salts in deionized water and two formulations of artificial saliva, at four temperatures and in the absence of agitation. Salt dissolution was quantified in vitro by analysis of video-microscopy images taken at different times. Higher dissolution rates of salt crystals were found in water than in artificial saliva, and at higher temperatures. Video-microscopy was instrumental to reveal that some crystals were fragmented during dissolution while others remained as a unit of decreasing size until complete solubilization. Increased surface area after fragmentation led to pyramid-shape crystals having the highest dissolution rates. Hence, significant changes can be achieved in the dissolution of salt depending on the crystal structure and its dissolution pattern.

Photochemical stability and reactivity of graphene oxide

The photoreactivity of graphene oxide (GO) suspensions was investigated with a double aim: (i) to give insights into the previously reported photo-reduction process, which allows a partial elimination of the oxygen-containing groups from the 2D graphitic structure; (ii) to explore the possible use of GO as photo-activator able to promote the photo-transformation/abatement of organic molecules. To reach these goals and clarify some peculiar aspects of the photochemistry of GO till now obscure or confuse, we synthesized and characterized stable GO suspensions which were then subjected to UV–Vis irradiation for prolonged times. GO underwent partial photo-reduction with the release of gaseous molecules and soluble organic species (e.g., carboxylic acid). The mechanisms of photo-reduction occurring under air or N2 are different, as assessed by the release in solution of diverse soluble molecules. In the presence of oxygen, at long irradiation time, a complete solubilization of the graphenic structures was observed. No difference in the nature and amount of released gases (principally CO2 and CO) was observed in the oxic or anoxic conditions. The possible use of GO as photo-activator was evaluated using phenol as probe molecule. GO revealed a double role of photo-activator and reagent in phenol degradation, as competition was assessed between GO self-transformation/reduction and phenol degradation. At prolonged irradiation time, a marked reactivity of the photoformed species was observed and the complete degradation was achieved for both organic small molecules formed from GO and the phenol added as probe molecule.

Evaluation of Heavy Metals Contamination from Environment to Food Matrix by TXRF: The Case of Rice and Rice Husk

This paper is devoted to the chemical analysis of contaminated soils of India and the rice grown in the same area. Total reflection X-ray fluorescence spectroscopy is a well-established technique for elemental chemical analysis of environmental samples, and it can be a useful tool to assess food safety. Metals uptake in rice crop grown in soils from different areas was studied. In this work soil, rice husk and rice samples were analyzed after complete solubilization of samples by microwave acid digestion. Heavy metals concentration detected in rice samples decreases in the following order: Mn > Zn > Cu > Ni > Pb > Cr. The metal content in rice husk was higher than in rice. This study suggests, for the first time, a possible role of heavy metals filter played by rice husk. The knowledge of metals sequestration capability of rice husk may promote some new management practices for rice cultivation to preserve it from pollution.

Formulation and In-vitro Evaluation of Sublingual Tablets of Ondansetron Hydrochloride using Coprocessed Excipients

Abstract: The formulation of sublingual tablets of Ondansetron HCl was carried out by using direct compression technique and evaluation tests were carried out as per pharmacopoeial specifications. Poor compressibility problem of Mannitol was overcome by coprocessing it with maltose and corn starch in varying ratios. The results of evaluation tests indicate that the ratio of Mannitol: Maltose: Corn starch: 19:2:1 gave better tableting performance with respect to precompression & postcompression parameters. It was also observed that increase in maltose content, increased the hardness but negatively affects disintegration and drug release and vice versa. Furthermore the study on effect of superdisintgrants shows that Crospovidone gives faster disintegration and satisfactory drug release in concentration of 4% compared to that of Sodium starch glycolate & Croscarmelose sodium. Formulation using a bioadhesive polymer PVP K 30 in ratio of 0.5% showed uniform release of drug over a period of 20 minutes with complete solubilization of tablet compared to that of gelatin and carbopol 934. On numerical optimization of prepared formulations, three formulations were suggested by Design Expert 8.0.7.1(Trial Version), among that Formulation B gave better correlation between predicted value and observed value. Thus Formulation B was chosen as global best formulation. Key words: Sublingual Tablet, Direct Compression, Co-processing of excipients, Historical Data Optimization, Ondansetron HCl.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

Fractionation Behavior of Metals (Al, Ni, V, and Mo) During Bioleaching and Chemical Leaching of Spent Petroleum Refinery Catalyst

We analyzed, for the first time, the different fractions of metals present in the spent catalyst and changes they undergone during bioleaching and chemical leaching. Before bioleaching, Al (83.9 %) was found mainly in the residual fraction of the pretreated spent catalyst. Ni (61.3 %) was mainly present in the exchangeable fraction exhibiting its high environmental mobility. V (58.5) and Mo (49.3 %) mainly existed in the oxidizable fraction suggesting that highly oxidizing conditions would liberate these metals from the spent catalyst. During bioleaching with Acidithiobacillus thiooxidans, almost complete solubilization of the exchangeable as well as of reducible fraction was observed. Due to strong acidic conditions, part of oxidizable fraction of these metals was also solubilized. Bioleaching also affected the fractionation of metals remaining in the treated spent catalyst. At the end of the process, most of the metals remaining in the spent catalyst were found in the more stable fractions ensuring the safe disposal of spent catalyst. The leaching yields and fractionation behavior of metals during chemical leaching was found to be identical. The results of the present study strongly suggest that bioleaching is an effective method for removing metals from the spent catalyst.

Quantitative proteomics in resected renal cancer tissue for biomarker discovery and profiling

Background:Proteomics-based approaches for biomarker discovery are promising strategies used in cancer research. We present state-of-art label-free quantitative proteomics method to assess proteome of renal cell carcinoma (RCC) compared with noncancer renal tissues.Methods:Fresh frozen tissue samples from eight primary RCC lesions and autologous adjacent normal renal tissues were obtained from surgically resected tumour-bearing kidneys. Proteins were extracted by complete solubilisation of tissues using filter-aided sample preparation (FASP) method. Trypsin digested proteins were analysed using quantitative label-free proteomics approach followed by data interpretation and pathways analysis.Results:A total of 1761 proteins were identified and quantified with high confidence (MASCOT ion score threshold of 35 and P-value <0.05). Of these, 596 proteins were identified as differentially expressed between cancer and noncancer tissues. Two upregulated proteins in tumour samples (adipose differentiation-related protein and Coronin 1A) were further validated by immunohistochemistry. Pathway analysis using IPA, KOBAS 2.0, DAVID functional annotation and FLink tools showed enrichment of many cancer-related biological processes and pathways such as oxidative phosphorylation, glycolysis and amino acid synthetic pathways.Conclusions:Our study identified a number of differentially expressed proteins and pathways using label-free proteomics approach in RCC compared with normal tissue samples. Two proteins validated in this study are the focus of on-going research in a large cohort of patients.

Are ionic liquids extremophiles cell wall breakers? Esther Gutiérrez, M. Ángeles Sanromán, Ana Rodríguez, Francisco J. Deive * Department of Chemical Engineering, University of Vigo, 36310, Vigo, Spain * Corresponding author: Tel.: +34986818723; E-mail address: deive@uvigo.es

Are ionic liquids extremophiles cell wall breakers? Esther Gutiérrez, M. Ángeles Sanromán, Ana Rodríguez, Francisco J. Deive * Department of Chemical Engineering, University of Vigo, 36310, Vigo, Spain * Corresponding author: Tel.: +34986818723; E-mail address: deive@uvigo.es

Utilizing Dynamic Light Scattering as a Process Analytical Technology for Protein Folding and Aggregation Monitoring in Vaccine Manufacturing

Protein aggregation is a common challenge in the manufacturing of biological products. It is possible to minimize the extent of aggregation through timely measurement and in-depth characterization of aggregation. In this study, we demonstrated the use of dynamic light scattering (DLS) to monitor inclusion body (IB) solubilization, protein refolding, and aggregation near the production line of a recombinant protein-based vaccine candidate. Our results were in good agreement with those measured by size-exclusion chromatography. DLS was also used to characterize the mechanism of aggregation. As DLS is a quick, nonperturbing technology, it can potentially be used as an at-line process analytical technology to ensure complete IB solubilization and aggregate-free refolding.

Functional Reconstitution of Staphylococcus aureus Truncated AgrC Histidine Kinase in a Model Membrane System

The integral membrane protein AgrC is a histidine kinase whose sensor domains interact with an autoinducing peptide, resulting in a series of downstream responses. In this study, truncated AgrCTM5-6C and AgrCTM5-6C-GFP with GFP as a reporter gene were produced using a bacterial system. Purified AgrCTM5-6C and AgrCTM5-6C-GFP were reconstituted into liposomes by a detergent-mediated method. To achieve high-yield protein incorporation, we investigated the effect of different detergents on protein reconstitution efficiency. The highest incorporation was found with N,N-dimethyldode-cylamine N-oxide during complete liposome solubilization, which resulted in a yield of 85±5%. The COOH-terminus of the protein AgrCTM5-6C was almost exclusively oriented towards the inside of the vesicles. AgrCTM5-6C in proteoliposomes exhibited approximately a 6-fold increase in constitutive activity compared with AgrCTM5-6C in detergent micelles. The reconstitution of AgrCTM5-6C or AgrCTM5-6C-GFP was characterized using dynamic light scattering, fluorescence microscopy, and transmission electron microscopy. Based on the results, the optimal conditions for protein incorporation were defined. These findings contribute to the study of membrane protein structure and function in vitro using a reconstitution system.

Abstract 282: Phospholamban Truncation Mutations Including Heart Failure Mutant L39stop Disrupt Membrane Localization.

Introduction: Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca 2+ handling and contractility by reversibly inhibiting SR Ca 2+ ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization. Hypothesis: We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation. Results and Methods: To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization. Conclusions: Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.