Judges’ Queries and Presenter’s Replies

  • Icon for: Jon Kellar

    Jon Kellar

    Judge
    May 20, 2013 | 11:51 a.m.

    How does cholesterol activity relate to cholersterol concentration?

  • Icon for: Benjamin Johnson

    Benjamin Johnson

    Presenter
    May 21, 2013 | 07:23 p.m.

    Cholesterol activity represents the actually chemical potential of cholesterol. It is related to cholesterol concentration by an activity coefficient that represents the deviation from an ideal interaction. Cholesterol molecules interact with the other membrane constituents that lower its ability to interact with other molecule and there by lowers the cholesterols chemical potential. The exact activity coefficient is determined by how strongly the other membrane constituents interact with cholesterol. A thermodynamic equation could be used to calculate the cholesterol activity of simple systems, but for something like a cell membrane, a probe such as ours would be required.

  • Icon for: Marc Porter

    Marc Porter

    Judge
    May 20, 2013 | 11:59 a.m.

    What is the basis for the study of temperature dependence in Panel 2 and what do the differences in the three sets of tests tell us?

  • Icon for: Benjamin Johnson

    Benjamin Johnson

    Presenter
    May 21, 2013 | 07:23 p.m.

    There is a kinetic barrier for the transition from the pre-pore to pore state. PFO does not actually transition while at 4 degree C, but rather will spontaneously insert very quickly in the membrane if the temperature is raised even slightly. The mutations we have made to PFO have caused the kinetic barrier for pore formation to be raised drastically and make pore formation very unlikely. This is due to the fact that these residues form hydrogen bonds in the transition and final state which lower the free energy of the system.

  • May 20, 2013 | 03:52 p.m.

    Is cholesterol activity related to the various types of cholesterol (HDL, LDL, VLDL)? If so, what differences would be expected in the interaction of PFO with these various types of cholesterol?

  • Icon for: Benjamin Johnson

    Benjamin Johnson

    Presenter
    May 21, 2013 | 07:22 p.m.

    HDL and LDL are transport proteins that solubilize fats particularly cholesterol for aqueous transport in the blood stream. PFO binds to the cholesterol molecules in the cell membrane. PFO would not bind to any of the lipoproteins, but cholesterol activity probably affects the binding of lipoproteins to membrane cholesterol. This is something that could be investigated with our probe.

  • May 21, 2013 | 07:02 a.m.

    What was the rationale for the D434S mutation?

  • Icon for: Benjamin Johnson

    Benjamin Johnson

    Presenter
    May 21, 2013 | 07:22 p.m.

    The goal of this mutation was to lower the amount of cholesterol the protein requires for binding. We choose D434S because it was shown to reside in the membrane upon binding and unpaired charges in the membrane are very unfavorable. We changed the residue to a serine because its hydrophobic nature would lower the energy of binding. We also selected this residue because it was not well conserved in the CDC family and mutating conserved residues, in our experience, usually has negative effects on the level of cholesterol required for probe binding.

  • Icon for: Antal Jakli

    Antal Jakli

    Judge
    May 22, 2013 | 09:28 p.m.

    Can you tell me how cholesterol activity is related to HDL and LDL cholesterols?

  • Icon for: Benjamin Johnson

    Benjamin Johnson

    Presenter
    May 22, 2013 | 11:12 p.m.

    LDL and HDL are transport protein for cholesterol and are used as a representative of a person’s overall cholesterol level. Cholesterol activity refers to cholesterol in given cell membrane and how a cells affective concentration of cholesterol is affect by the other membrane constituents. Cholesterol activity is related to cholesterol concentration by an activity coefficient that represents the deviation from an ideal interaction. LDL and HDL are both up regulated in response to high cholesterol concentration in the blood stream, especially LDL. These lipoproteins will increase the transport of cholesterol to the cells and raise their cholesterol concentration. Cells though regulate their cholesterol activity very tightly and in the face of increased cholesterol concentration they can modify the other membrane constituents to accommodate the increased concentration of cholesterol. This will result in minimal changes in cholesterol activity except in extreme cases.

  • Further posting is closed as the competition has ended.

Presentation Discussion

  • Icon for: Brian Drayton

    Brian Drayton

    Faculty
    May 23, 2013 | 11:15 p.m.

    Hi,
    My biochemistry is basic: I am not familiar with the term “trafficking” — does this mean the rate of cholesterol molecules (or components) moving across the membranes? Not clear about the importance of the contrast with concentrations of cholesterols.

  • Further posting is closed as the competition has ended.

Icon for: Benjamin Johnson

BENJAMIN JOHNSON

University of Massachusetts at Amherst
Years in Grad School: 5

DETECTING CHANGES IN CHOLESTEROL ACTIVITY (ACCESSIBILITY) AT THE MEMBRANE SURFACE USING PERFRINGOLYSIN O MUTANTS

Cholesterol is an essential component of mammalian cell membranes and it is important to regulate the structure and function of lipid bilayers. Changes in cholesterol levels are involved in many physiological and pathological events such as the formation of arterial plaques, viral entry into cells, sperm capacitation, and receptor organization. Determination of cholesterol trafficking and distribution is essential for understanding how cells regulate cholesterol activity. A cholesterol probe capable of distinguishing changes in cholesterol chemical activity within membranes would facilitate investigations in this area. Perfringolysin O (PFO) is a cytolysin secreted by Clostridium perfringens that requires cholesterol in the target cell membrane for binding. The specificity of PFO for high levels of active cholesterol makes the toxin a potential tool for the detection of cholesterol distribution and trafficking. In an effort to adapt PFO into a molecular probe capable of sensing changes in membrane cholesterol activity, we have taken a non-lytic derivative of PFO and introduced several point mutations in the membrane-interacting domain 4 loops. These mutations altered the threshold of cholesterol concentration required in the membrane to trigger binding . The cholesterol-dependent binding of each PFO derivative was tested on model membranes containing different amounts of cholesterol. Three PFO derivatives were selected to test their binding to macrophage plasma membranes. These three derivatives showed differential binding to cell membranes treated with ?-methyl-cyclodextrin/cholesterol mixtures. Our data showed that the produced PFO derivatives differentially bind to model and biological membranes containing different cholesterol activity (or accessibility).