Nascent clathrin lattices spontaneously disassemble without sufficient adaptor proteins

Clathrin-coated structures must assemble on cell membranes to perform their primary function of receptor internalization. These structures show marked plasticity and instability, but what conditions are necessary to stabilize against disassembly have not been quantified. Recent in vitro fluorescence experiments have measured kinetics of stable clathrin assembly on membranes as controlled by key adaptor proteins like AP-2. Here, we combine this experimental data with microscopic reaction-diffusion simulations and theory to quantify mechanisms of stable vs unstable clathrin assembly on membranes. Both adaptor binding and dimensional reduction on the 2D surface are necessary to reproduce the cooperative kinetics of assembly. By applying our model to more physiologic-like conditions, where the stoichiometry and volume to area ratio are significantly lower than in vitro, we show that the critical nucleus contains ~25 clathrin, remarkably similar to sizes of abortive structures observed in vivo. Stable nucleation requires a stoichiometry of adaptor to clathrin that exceeds 1:1, meaning that AP-2 on its own has too few copies to nucleate lattices. Increasing adaptor concentration increases lattice sizes and nucleation speeds. For curved clathrin cages, we quantify both the cost of bending the membrane and the stabilization required to nucleate cages in solution. We find the energetics are comparable, suggesting that curving the lattice could offset the bending energy cost. Our model predicts how adaptor density controls stabilization of clathrin-coated structures against spontaneous disassembly, and shows remodeling and disassembly does not require ATPases, which is a critical advance towards predicting control of productive vesicle formation.

Complex Reaction Kinetics in Chemistry: A Unified Picture Suggested by Mechanics in Physics

Complex biochemical pathways can be reduced to chains of elementary reactions, which can be described in terms of chemical kinetics. Among the elementary reactions so far extensively investigated, we recall the Michaelis-Menten and the Hill positive-cooperative kinetics, which apply to molecular binding and are characterized by the absence and the presence, respectively, of cooperative interactions between binding sites. However, there is evidence of reactions displaying a more complex pattern: these follow the positive-cooperative scenario at small substrate concentration, yet negative-cooperative effects emerge as the substrate concentration is increased. Here, we analyze the formal analogy between the mathematical backbone of (classical) reaction kinetics in Chemistry and that of (classical) mechanics in Physics. We first show that standard cooperative kinetics can be framed in terms of classical mechanics, where the emerging phenomenology can be obtained by applying the principle of least action of classical mechanics. Further, since the saturation function plays in Chemistry the same role played by velocity in Physics, we show that a relativistic scaffold naturally accounts for the kinetics of the above-mentioned complex reactions. The proposed formalism yields to a unique, consistent picture for cooperative-like reactions and to a stronger mathematical control.

Cooperative kinetics of depolarization in CH3NH3PbI3 perovskite solar cells

Power law voltage decay in perovskite solar cells shows cooperative relaxation phenomena.

Kinetic Characterisation of Phosphofructokinase Purified from Setaria cervi: A Bovine Filarial Parasite

Phosphofructokinase (PFK), a regulatory enzyme in glycolytic pathway, has been purified to electrophoretic homogeneity from adult female Setaria cervi and partially characterized. For this enzyme, the Lineweaver-Burk's double reciprocal plots of initial rates and D-fructose-6-phosphate (F-6-P) or Mg-ATP concentrations for varying values of cosubstrate concentration gave intersecting lines indicating that Km values for F-6-P (1.05 mM) and ATP (3 μM) were independent of each other. S. cervi PFK, when assayed at inhibitory concentration of ATP (>0.1 mM), exhibited sigmoidal behavior towards binding with F-6-P with a Hill coefficient (n) value equal to 1.8 and 1.7 at 1.0 and 0.33 mM ATP, respectively. D-fructose-1,6-diphosphate (FDP) competitively inhibited the filarial enzyme: Ki and Hill coefficient values being 0.18 μM and 2.0, respectively. Phosphoenolpyruvate (PEP) also inhibited the enzyme competitively with the Ki value equal to 0.8 mM. The Hill coefficient values (>1.5) for F-6-P (at inhibitory concentration of ATP) and FDP suggested its positive cooperative kinetics towards F-6-P and FDP, showing presence of more than one binding sites for these molecules in enzyme protein and allosteric nature of the filarial enzyme. The product inhibition studies gave us the only compatible mechanism of random addition process with a probable orientation of substrates and products on the enzyme surface.

Biochemical Characteristics of the Novel Haloalkane Dehalogenase DatA, Isolated from the Plant PathogenAgrobacterium tumefaciensC58

ABSTRACTWe report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogenAgrobacterium tumefaciensC58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene

The phosphofructokinase from the non-conventional yeast Yarrowia lipolytica (YlPfk) was purified to homogeneity, and its encoding gene isolated. YlPfk is an octamer of 869 kDa composed of a single type of subunit, and shows atypical kinetic characteristics. It did not exhibit cooperative kinetics for fructose 6-phosphate (Hill coefficient, h 1·1; S 0·5 52 μM), it was inhibited moderately by MgATP (K i 3·5 mM), and it was strongly inhibited by phosphoenolpyruvate (K i 61 μM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The gene YlPFK1 has no introns, and encodes a putative protein of 953 aa, with a molecular mass consistent with the subunit size found after purification. Disruption of the gene abolished growth in glucose and Pfk activity, while reintroduction of the gene restored both properties. This indicates that Y. lipolytica has only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of the Ylpfk1 disruptant in permissive carbon sources. The unusual kinetic characteristics of YlPfk, and the intracellular concentrations of glycolytic intermediates during growth in glucose, suggest that YlPfk may play an important role in the regulation of glucose metabolism in Y. lipolytica, different from the role played by the enzyme in Saccharomyces cerevisiae.