Anne Hinderliter

Stability of protein-decorated mixed lipid membranes: The interplay of lipid-lipid, lipid-protein, and protein-protein interactions.

Membrane-associated proteins are likely to contribute to the regulation of the phase behavior of mixed lipid membranes. To gain insight into the underlying mechanism, we study a thermodynamic model for the stability of a protein-decorated binary lipid layer. Here, proteins interact preferentially with one lipid species and thus locally sequester that species. We aim to specify conditions that lead to an additional macroscopic phase separation of the protein-decorated lipid membrane. Our model is based on a standard mean-field lattice-gas description for both the lipid mixture and the adsorbed protein layer. Besides accounting for the lipid-protein binding strength, we also include attractive lipid-lipid and protein-protein interactions. Our analysis characterizes the decrease in the membranes critical interaction parameter as a function of the lipid-protein binding strength. For small and large binding strengths we provide analytical expressions; numerical results cover the intermediate range. Our results reiterate the crucial importance of the line tension associated with protein-induced compositional gradients and the presence of attractive lipid-lipid interactions within the membrane. Direct protein-protein attraction effectively increases the line tension and thus tends to further destabilize the membrane.

Peripheral Protein Organization and Its Influence on Lipid Diffusion in Biomimetic Membranes

Protein organization on biomembranes and their dynamics are essential for cellular function. It is not clear, however, how protein binding may influence the assembly of underlying lipids or how the membrane structure leads to functional protein organization. Toward this goal, we investigated the effects of annexin a5 binding to biomimetic membranes using fluorescence imaging and correlation spectroscopy. Annexin a5 (anx a5), a peripheral intracellular protein that plays a membrane remodeling role in addition to other functions, binds specifically and tightly to anionic (e.g., phosphatidylserine)-containing membranes in the presence of calcium ion. Our fluorescence microscopy reveals that annexin likely forms assemblies, along with a more dispersed population, upon binding to anionic biomembranes in the presence of calcium ion, which is reflected in its two-component Brownian motion. To investigate the effects of annexin binding on the underlying lipids, we used specific acyl chain labeled phospholipid analogues, NBD-phosphatidylcholine (NBD-PC) and NBD-phosphatidylserine (NBD-PS). We find that both NBD-labeled lipids cluster under anx a5 assemblies, as compared with when they are found under the dispersed annexin population, and NBD-PS exhibits two-component lateral diffusion under the annexin assemblies. In contrast, NBD-PC diffusion is slower by an order of magnitude under the annexin assemblies in contrast to its diffusion when not localized under anx a5 assemblies. Our results indicate that, upon binding to membranes, the peripheral protein annexin organizes the underlying lipids into domains, which may have functional implications in vivo.

Mechanism for Calcium Ion Sensing by the C2A Domain of Synaptotagmin I

The C2A domain is one of two calcium ion (Ca(2+))- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca(2+) sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2As response to Ca(2+) and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca(2+)- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca(2+)- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.

Intra and Inter-Molecular Interactions Dictate the Aggregation State of Irinotecan Co-Encapsulated with Floxuridine Inside Liposomes

PurposeThe inter/intramolecular interactions between drugs (floxuridine, irinotecan) and excipients (copper gluconate, triethanolamine) in the dual-drug liposomal formulation CPX-1 were elucidated in order to identify the physicochemical properties that allow coordinated release of irinotecan and floxuridine and maintenance of the two agents at a fixed, synergistic 1:1 molar ratio.MethodsRelease of irinotecan and floxuridine from the liposomes was assessed using an in vitro-release assay. Fluorescence, Nuclear Magnetic Resonance spectroscopy (NMR) and UV–Vis were used to characterize the aggregation state of the drugs within the liposomes.ResultsCoordinated release of the drugs from liposomes was disrupted by removing copper gluconate. Approximately 45% of the total irinotecan was detectable in the copper-containing CPX-1 formulation by NMR, which decreased to 19% without copper present in the liposomal interior. Formation of higher order, NMR-silent aggregates was associated with slower and uncoordinated irinotecan release relative to floxuridine and loss of the synergistic drug/drug ratio. Solution spectroscopy and calorimetry revealed that while all formulation components were required to achieve the highest solubility of irinotecan, direct drug-excipient binding interactions were absent.ConclusionsLong-range interactions between irinotecan, floxuridine and excipients modulate the aggregation state of irinotecan, allowing for simultaneous release of both drugs from the liposomes.

Oxidation increases the strength of the methionine-aromatic interaction

Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we show that oxidation increases the strength of the methionine-aromatic interaction motif—a driving force for protein folding and protein-protein interaction—by 0.5 – 1.4 kcal/mol. We find that non-hydrogen bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups are enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR experiments on model peptides demonstrate that oxidation of methionine stabilizes the interaction by 0.5–0.6 kcal/mol. We confirm the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on 1) calmodulin structure and dynamics and 2) lymphotoxin-α/TNFR1 binding. Thus, the methionine-aromatic motif is a determinant of protein structural and functional sensitivity to oxidative stress.

Negative Coupling as a Mechanism for Signal Propagation between C2 Domains of Synaptotagmin I

Synaptotagmin I (Syt I) is a vesicle-localized protein implicated in sensing the calcium influx that triggers fast synchronous release of neurotransmitter. How Syt I utilizes its two C2 domains to integrate signals and mediate neurotransmission has continued to be a controversial area of research, though prevalent hypotheses favor independent function. Using differential scanning calorimetry and fluorescence lifetime spectroscopy in a thermodynamic denaturation approach, we tested an alternative hypothesis in which both domains interact to cooperatively disseminate binding information. The free energy of stability was determined for C2A, C2B, and C2AB constructs by globally fitting both methods to a two-state model of unfolding. By comparing the additive free energies of C2A and C2B with C2AB, we identified a negative coupling interaction between the C2 domains of Syt I. This interaction not only provides a mechanistic means for propagating signals, but also a possible means for coordinating the molecular events of neurotransmission.

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.

Monte Carlo simulation of protein-induced lipid demixing in a membrane with interactions derived from experiment.

Lipid 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 (Ca(2+)), 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 Ca(2+). 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.

Allostery and instability in the functional plasticity of synaptotagmin I

Synaptotagmin I (Syt I) is the calcium ion sensor for regulated release of neurotransmitter. How Syt I mediates this cellular event has been a question of extensive study for decades and yet, a clear understanding of the protein’s diverse functionality has remained elusive. Using tools of thermodynamics, we have identified two intrinsic properties that may account for Syt I’s functional plasticity: marginal stability and negative coupling. These two intrinsic properties have the potential to provide great conformational flexibility and suggest that Syt I’s functional plasticity stems in part from subtle rearrangements in the protein’s conformational ensemble. This model for Syt I function is discussed within the context of the nervous system’s overall plasticity.

Synaptotagmin I's Intrinsically Disordered Region Interacts with Synaptic Vesicle Lipids and Exerts Allosteric Control over C2A.

Synaptotagmin I (Syt I) is a vesicle-localized integral membrane protein that senses the calcium ion (Ca(2+)) influx to trigger fast synchronous release of neurotransmitter. How the cytosolic domains of Syt I allosterically communicate to propagate the Ca(2+) binding signal throughout the protein is not well understood. In particular, it is unclear whether the intrinsically disordered region (IDR) between Syt Is transmembrane helix and first C2 domain (C2A) plays an important role in allosteric modulation of Ca(2+) binding. Moreover, the structural propensity of this IDR with respect to membrane lipid composition is unknown. Using differential scanning and isothermal titration calorimetry, we found that inclusion of the IDR does indeed allosterically modulate Ca(2+) binding within the first C2 domain. Additionally through application of nuclear magnetic resonance, we found that Syt Is IDR interacts with membranes whose lipid composition mimics that of a synaptic vesicle. These findings not only indicate that Syt Is IDR plays a role in regulating Syt Is Ca(2+) sensing but also indicate the IDR is exquisitely sensitive to the underlying membrane lipids. The latter observation suggests the IDR is a key route for communication of lipid organization to the adjacent C2 domains.