Monte Carlo Simulation of Protein-Induced Lipid Demixing in a Membrane with Interactions Derived from Experiment

AbstractLipid domain formation induced by annexin was investigated in mixtures of phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (Chol), which were selected to mimic the inner leaflet of a eukaryotic plasma membrane. Annexins are ubiquitous and abundant cytoplasmic, peripheral proteins, which bind to membranes containing PS in the presence of calcium ions (Ca2+), but whose function is unknown. Prompted by indications of interplay between the presence of cholesterol in PS/PC mixtures and the binding of annexins, we used Monte Carlo simulations to investigate protein and lipid domain formation in these mixtures. The set of interaction parameters between lipids and proteins was assigned by matching experimental observables to corresponding variables in the calculations. In the case of monounsaturated phospholipids, the PS-PC and PC-Chol interactions are weakly repulsive. The interaction between protein and PS was determined based on experiments of annexin binding to PC/PS mixtures in the presence of Ca2+. Based on the proposal that PS and cholesterol form a complex in model membranes, a favorable PS-Chol interaction was postulated. Finally, protein-protein favorable interactions were also included, which are consistent with observations of large, two-dimensional, regular arrays of annexins on membranes. Those net interactions between pairs of lipids, proteins and lipids, and between proteins are all small, of the order of the average kinetic energy. We found that annexin a5 can induce formation of large PS domains, coincident with protein domains, but only if cholesterol is present

Development and Synthesis of Utrophin Actin Binding Domain 1 (ABD1)

University Honors Capstone Project and Poster, University of Minnesota Duluth, 2016. Kate McMahon authored paper and poster; Ben Horn, Dr. Jacob Gauer, and Dr. Anne Hinderliter authored poster.Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease containing point mutations in the muscle protein Dystrophin causing the protein to lose its function. Specifically, Dystrophin is critical for dissipating the mechanical stress placed on muscles during physical activity. Although Dystrophin is nonfunctional in DMD patients, its fetal homolog, Utrophin, is often present in higher amounts than common to adult cells. Because Utrophin and Dystrophin share 85% homology in their first actin binding domains (ABD1), the interrelatedness of structure and function validate Utrophin as a proposed therapeutic tool for combating DMD. To test this hypothesis, the thermodynamic character of Utrophin ABD1 and Dystrophin ABD1 will be compared. As Utrophin is not regularly studied, the gene for Utrophin ABD1 was designed, synthesized, and expressed in E.coli cells. Prokaryotic cells were utilized to express a eukaryotic protein because of rapid growth rate and the presence of an extra, self-replicating, circular DNA called a plasmid. A plasmid is evolutionarily advantageous because it can be passed quickly from prokaryotic cell to prokaryotic cell without the entire genome replicating, thus increasing variability. This unique attribute was utilized to express Utrophin ABD1 in E. coli cells. Although eukaryotic systems often have posttranslational modifications, this did not pose a threat for the prokaryotic cell amplification. The gene encoding the protein was designed using specific amino acid residues, not nucleotide sequences; the splicing of nucleotide sequences was irrelevant as posttranslational modification occurs before the amino acids are assembled into their primary structure. Specifically, Utrophin ABD1 was designed with BamHI and XhoI restriction enzymes flanking the 246 amino acid Utrophin ABD1 construct which was synthesized in a pUC57 E. coli plasmid. Using BamHI and XhoI, the amino acid sequence was restriction digested and subcloned into an expression vector containing components critical for nickel column chromatography like a histidine tag, TEV protease cut site, and maltose binding protein. The expression vector also contains a selective marker to find the correct ligated species such as the antibiotic Kanamycin. These plasmids were transformed into competent E. coli cells so the E. coli cells would replicate the inserted DNA the same way it replicates a plasmid. During the rapid growth, inclusion bodies, protein aggregates of overexpressed protein, are accounted for by the addition of the maltose binding protein which maintains solubility. The transformed cells were stored in a glycerol stock. Synthesis of this gene then allows growth and purification of the Utrophin ABD1 protein in a similar manner to those already classified for histidine tagged proteins. Purification is carried out at a pH of 8 so that the six histidines will be deprotonated and bind to the nickel column, thus washing out all other protein expect for the Utrophin bound to the column. Purification is important in that it insures pure protein by cleaving off the maltose binding protein using the tobacco etch virus (TEV) protease that recognizes a specific nucleotide sequence rarely found in the eukaryotic genome. Finally, thermodynamic analysis of this protein will give insight into the structure and function of Utrophin ABD1 and its potential capabilities as a therapeutic agent for patients with DMD

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response

Domain Formation in a Fluid Mixed Lipid Bilayer Modulated through Binding of the C2 Protein Motif

The role and mechanism of formation of lipid domains in a functional membrane have generally received limited attention. Our approach, based on the hypothesis that thermodynamic coupling between lipid−lipid and protein−lipid interactions can lead to domain formation, uses a combination of an experimental lipid bilayer model system and Monte Carlo computer simulations of a simple model of that system. The experimental system is a fluid bilayer composed of a binary mixture of phosphatidylcholine (PC) and phosphatidylserine (PS), containing 4% of a pyrene-labeled anionic phospholipid. Addition of the C2 protein motif (a structural domain found in proteins implicated in eukaryotic signal transduction and cellular trafficking processes) to the bilayer first increases and then decreases the excimer/monomer ratio of the pyrene fluorescence. We interpret this to mean that protein binding induces anionic lipid domain formation until the anionic lipid becomes saturated with protein. Monte Carlo simulations were performed on a lattice representing the lipid bilayer to which proteins were added. The important parameters are an unlike lipid−lipid interaction term and an experimentally derived preferential protein−lipid interaction term. The simulations support the experimental conclusion and indicate the existence of a maximum in PS domain size as a function of protein concentration. Thus, lipid−protein coupling is a possible mechanism for both lipid and protein clustering on a fluid bilayer. Such domains could be precursors of larger lipid−protein clusters (‘rafts'), which could be important in various biological processes such as signal transduction at the level of the cell membrane

Enhanced synaptotagmin plasticity derived from pairing intrinsic disorder with synaptic vesicle lipids

Synaptotagmin 1 (Syt 1) is an integral membrane protein responsible for sensing the calcium ion (Ca2+) influx in neurons that triggers synaptic vesicle exocytosis. How Syt 1's intrinsically disordered region (IDR), a ∼60 residue sequence located between the protein's transmembrane helix and two Ca2+-sensing C2 domains, contributes to protein function is not well understood. The same is true of analogous IDRs located in the other synaptotagmin isoforms. Recently, we found that the Syt 1 IDR is structurally responsive to vesicles whose lipid composition mimics that of a synaptic vesicle organelle and that this sensitivity allosterically influences binding and folding behavior of the adjacent C2 domain. We believe these observations may be applicable to the study of other synaptotagmin isoforms and discuss generally how an IDR-membrane interaction could contribute to modulation of C2 domain function