Estimating the Cholesterol Affinity of Integral Membrane Proteins from Experimental Data.

The cholesterol affinities of many integral plasma membrane proteins have been estimated by molecular computation. However, these values lack experimental confirmation. We therefore developed a simple mathematical model to extract sterol affinity constants and stoichiometries from published isotherms for the dependence of the activity of such proteins on the membrane cholesterol concentration. The binding curves for these proteins are sigmoidal, with strongly lagged thresholds attributable to competition for the cholesterol by bilayer phospholipids. The model provided isotherms that matched the experimental data using published values for the sterol association constants and stoichiometries of the phospholipids. Three oligomeric transporters were found to bind cholesterol without cooperativity, with dimensionless association constants of 35 for Kir3.4* and 100 for both Kir2 and a GAT transporter. (The corresponding ΔG° values were -8.8, -11.4, and -11.4 kJ/mol, respectively). These association constants are significantly lower than those for the phospholipids, which range from ∼100 to 6000. The BK channel, the nicotinic acetylcholine receptor, and the M192I mutant of Kir3.4* appear to bind multiple cholesterol molecules cooperatively (n = 2 or 4), with subunit affinities of 563, 950, and 700, respectively. The model predicts that the three less avid transporters are approximately half-saturated in their native plasma membranes; hence, they are sensitive to variations in cholesterol in vivo. The more avid proteins would be nearly saturated in vivo. The method can be applied to any integral protein or other ligands in any bilayer for which there are reasonable estimates of the sterol affinities and stoichiometries of the phospholipids.

Microtubule orientation and spacing within bundles is critical for long-range kinesin-1 motility.

Cells rely on active transport to quickly organize cellular cargo. How cells regulate transport is not fully understood. One proposed mechanism is that motor activity could be altered through the architecture of the cytoskeleton. This mechanism is supported by the fact that the cytoskeletal network is tightly regulated in cells and filament polarity within networks dictates motor directionality. For instance, axons contain bundles of parallel microtubules and all cargos with the same motor species will move in the same direction. It is not clear how other types of networks, such as antiparallel bundles in dendrites, can regulate motor transport. To understand how the organization of microtubules within bundles can regulate transport, we studied kinesin-1 motility on three bundle types: random-polarity bundles that are close-packed, parallel polarity bundles, and antiparallel polarity bundles that are spaced apart. We find that close-packed bundles inhibit motor motion, while parallel arrays support unidirectional motion. Spacing the microtubules with microtubule-associated proteins enhances run lengths. Our results indicate that microtubule bundle architecture dictates the motion of single motors and could have effects on cargo transport. © 2014 Wiley Periodicals, Inc.

Model for competition from self during passive immunization, with application to broadly neutralizing antibodies for HIV.

We propose a mathematical model to interpret observations concerning the behavior of broadly neutralizing antibodies for chronic HIV in vivo. The model enables us to identify a threshold antibody level that must be achieved to decrease the viral load effectively. Although this threshold has not been reached in existing passive immunization studies, it is within range of humoral immune responses, suggesting that therapeutic vaccines are feasible. In an appendix, we develop a model of passive immunization against influenza, and acute infection.