Peptides derived from MARCKS block coagulation complex assembly on phosphatidylserine.

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi

Effects of Membrane Shape and Lipid Composition in Extracellular Vesicle and Platelet Biology

In this work, I examine the importance of fundamental properties of lipid membranes, such as membrane curvature or lipid composition, in the context of extracellular vesicle and platelet biology. Although differing in biologic function, both extracellular vesicles and platelets are comparatively small in size, anucleate, and expose phosphatidylserine on their outer membrane leaflet. Phosphatidylserine is an anionic lipid that is generally sequestered to the inner leaflet of bilayer membranes. The exposure of phosphatidylserine on the outer membrane leaflet of extracellular vesicles appears necessary for their signaling, and the exposure of phosphatidylserine on platelets facilitates the assembly of enzymatically active coagulation protein complexes. I first highlight the basic biology of extracellular vesicles and address biochemical and biophysical detection methods that depend on the lipid composition and particle size of the extracellular vesicles. Next, I use all-atom molecular dynamics simulations to show that increasing the lateral density of lipids can induce a bilayer membrane to form a curved shape. These membranes provide a model system for studying interactions in curved membranes, as well as demonstrate that curved membranes display an increased number of lipid packing defects. Next, I adapt theoretical models of membrane-membrane interactions to examine the interaction energies between extracellular vesicles and cells. These estimates show that smaller vesicles such as exosomes are more likely to signal via endocytosis, while larger vesicles like microvesicles are more likely to signal via receptor-ligand interactions. Finally, I examine the effects of phosphatidylserine-targeting peptides on the platelet procoagulant response. I show that these peptides can compete with coagulation factors for phosphatidylserine binding sites and target phosphatidylserine exposed on activated platelets in vitro and in vivo. Together, this work supports a broader understanding of how membrane shape and lipid composition influences, and is a potential target for modulation of, the biology of extracellular vesicles and platelets

Ice–Liquid Oscillations in Nanoconfined Water

Nanoscale confinement has a strong effect on the phase behavior of water. Studies in the last two decades have revealed a wealth of novel crystalline and quasicrystalline structures for water confined in nanoslits. Less is known, however, about the nature of ice–liquid coexistence in extremely nanoconfined systems. Here, we use molecular simulations to investigate the ice–liquid equilibrium for water confined between two nanoscopic disks. We find that the nature of ice–liquid phase coexistence in nanoconfined water is different from coexistence in both bulk water and extended nanoslits. In highly nanoconfined systems, liquid water and ice do not coexist in space because the two-phase states are unstable. The confined ice and liquid phases coexist in time, through oscillations between all-liquid and all-crystalline states. The avoidance of spatial coexistence of ice and liquid originates on the non-negligible cost of the interface between confined ice and liquid in a small system. It is the result of the small number of water molecules between the plates and has no analogue in bulk water

Vapor deposition of water on graphitic surfaces: formation of amorphous ice, bilayer ice, ice I, and liquid water

Carbonaceous surfaces are a major source of atmospheric particles and could play an important role in the formation of ice. Here we investigate through molecular simulations the stability, metastability, and molecular pathways of deposition of amorphous ice, bilayer ice, and ice I from water vapor on graphitic and atomless Lennard-Jones surfaces as a function of temperature. We find that bilayer ice is the most stable ice polymorph for small cluster sizes, nevertheless it can grow metastable well above its region of thermodynamic stability. In agreement with experiments, the simulations predict that on increasing temperature the outcome of water deposition is amorphous ice, bilayer ice, ice I, and liquid water. The deposition nucleation of bilayer ice and ice I is preceded by the formation of small liquid clusters, which have two wetting states: bilayer pancake-like (wetting) at small cluster size and droplet-like (non-wetting) at larger cluster size. The wetting state of liquid clusters determines which ice polymorph is nucleated: bilayer ice nucleates from wetting bilayer liquid clusters and ice I from non-wetting liquid clusters. The maximum temperature for nucleation of bilayer ice on flat surfaces, T(B)(max) is given by the maximum temperature for which liquid water clusters reach the equilibrium melting line of bilayer ice as wetting bilayer clusters. Increasing water-surface attraction stabilizes the pancake-like wetting state of liquid clusters leading to larger T(B)(max) for the flat non-hydrogen bonding surfaces of this study. The findings of this study should be of relevance for the understanding of ice formation by deposition mode on carbonaceous atmospheric particles, including soot

Ice–Liquid Oscillations in Nanoconfined Water

Nanoscale confinement has a strong effect on the phase behavior of water. Studies in the last two decades have revealed a wealth of novel crystalline and quasicrystalline structures for water confined in nanoslits. Less is known, however, about the nature of ice–liquid coexistence in extremely nanoconfined systems. Here, we use molecular simulations to investigate the ice–liquid equilibrium for water confined between two nanoscopic disks. We find that the nature of ice–liquid phase coexistence in nanoconfined water is different from coexistence in both bulk water and extended nanoslits. In highly nanoconfined systems, liquid water and ice do not coexist in space because the two-phase states are unstable. The confined ice and liquid phases coexist in time, through oscillations between all-liquid and all-crystalline states. The avoidance of spatial coexistence of ice and liquid originates on the non-negligible cost of the interface between confined ice and liquid in a small system. It is the result of the small number of water molecules between the plates and has no analogue in bulk water

Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms.

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions

Evaluating the potential for epistasis.

<p>(A) The number of residues in each motif are determined for all representative thermophilic-mesophilic structure pairs and binned according to the motif size. (B) The number of residue substitutions, given as Hamming distance, in each equivalent thermophilic-mesophilic motif is determined and binned.</p

The backbone can move significantly in the structurally equivalent clusters.

<p>(A) Three Cα atoms from a paired cluster are shown in red spheres (thermophilic enzyme) and purple spheres (mesophilic enzyme). The atoms are labeled a, b and c for the thermophilic enzyme and a’, b’ and c’ for the mesophilic enzyme. The Euclidian distances between Cα atoms are shown for each enzyme, with the distance differences at right. (B) The sum of the absolute values for the distance differences (red), and the average distance differences (blue) for each representative cluster are shown, sorted by summed or averaged distances.</p

Thermophilic enzyme clusters display closer atomic packing compared to mesophilic enzyme clusters for most enzyme pairs evaluated.

<p>(A) SASA_1.4 values for clusters from the representative thermophilic-mesophilic structure pairs are shown, with thermophilic clusters shown in red, mesophilic clusters in green and the difference, ΔSASA_1.4, in blue. Values are sorted by ΔSASA_1.4. (B) SASA_1.4 values are shown comparing clusters from the thermophilic (PDB 1a5z) and mesophilic (PDB 6ldh) lactate dehydrogenase enzymes, which have a difference in optimum activity temperature of 30°C. (C) the thermophilic (PDB 1a5z) and mesophilic (PDB 5ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 48°C, (D) and the thermophilic (PDB 1a5z) and psychrophilic (PDB 1ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 70°C.</p

Anchor residues are conserved in atomic packing.

<p>(A) The thermostable GH9 (PDB 4dod) is shown in surface representation, with anchor residues that are seen in a larger number of clusters shown in stick representation. Residues exhibiting the largest ΔSASA_1.4, which are never anchor residues, are colored red. (B) Sequence positions from 4dod are binned by the number of clusters in which they are found. The heat scale indicates ΔSASA_1.4. Importantly, blue is not observed as there are no mesophilic clusters with significantly better atomic packing relative to the matched thermophilic cluster. (C) Sequence positions from the representative set of structures are binned by the number of motifs in which they are found (x-axis), with ΔSASA_1.4 shown for each paired position (y-axis). A white symbol indicates sequence conservation, and gray indicates the sequence differs at that position.</p