Ryan Mahling

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.

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.

Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cells ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

The Protein that Held Back the Dye: Annexin's Effect on Membrane Permeability

The membrane is more than a barrier that protects the cell; composed of lipids and protein, the membrane is implicated in signaling, cell stability, and protein interactions. They must be able to respond to stressors that can affect these roles, and employ different membrane components for that purpose. Cholesterol is a common component to both monolayers of the eukaryotic plasma membrane, moving freely and relaying of information such as changes in lipid distribution. The annexin family of membrane-associated proteins constitutes two percent of eukaryotic proteins within the cell. Annexins interact with multiple binding partners including small molecules like Ca2+, phospholipids, and other proteins that are often involved in membrane repair. To determine how binding of annexin impacts the permeability of the membrane, carboxyfluorescein (CF) release assays were performed. CF efflux from vesicles in the presence of annexin a5 without Ca2+ showed a slight decrease compared to the control of vesicles alone; however, with the addition of both annexin and Ca2+, the signal decrease was greater. In order to observe the effects of both cholesterol and protein on permeability, CF studies were repeated on vesicles containing increasing mole fractions of cholesterol. Less CF was released from vesicles containing cholesterol, and an even greater decrease was observed with annexin and Ca2+ added. This suggests that in the presence of Ca2+, annexin works to reduce the permeability of the membrane, especially for cholesterol-containing vesicles. In previous work with isothermal titration calorimetry (ITC) we also observed a change in the Ca2+ binding parameters and stoichiometry of annexin a5 in the presence of cholesterol-containing membranes. This combined data, leads us to hypothesize that through their calcium binding ability, annexins sense the distribution of lipids and help communicate changes in the membrane environment.

Membranes in Flux: Proteins Effect on Membrane Permeability

Lipid membranes are the basis of cell structure by providing stability and regulation against certain stressors. The permeability of this membrane is regulated through the distribution of phospholipids across a lipid bilayer. With the addition of cholesterol to the lipid bilayer the mixing behaviors of the phospholipids present in the membranes are altered causing fluctuation in permeability. Cholesterol has the ability to move freely across monolayers, contributing to the communication passing through the bilayer. Annexin and the C2A domain of Synaptotagmin 1 are two types of membrane binding proteins whose binding affinity is affected in the presence of calcium ions (Ca2+). With the combination of cholesterol containing lipid bilayers, Ca2+, and protein the distribution of phospholipids and cholesterol is fluctuated, causing alteration in the membranes permeability. Both proteins were used in a carboxyfluorescein (CF) release assay to study the fluctuation in membrane permeability with increasing concentrations of protein. The measured efflux of this fluorescent dye in the presence of increasing concentrations of protein with and without Ca2+ shows these fluctuations in permeability across the lipid membrane. The amount of cholesterol present in the membrane correlates with the measured CF efflux released through the lipid bilayer upon binding of protein and Ca2+. We hypothesize that membrane binding proteins have the ability to sense certain distributions of phospholipids and cholesterol across the lipid membrane and communicate changes within the membrane environment.