Quantitation of Electrostatic and Hydrophobic Membrane Interactions by Equilibrium Dialysis and Reverse-Phase HPLC

Equilibrium dialysis and reverse-phase HPLC have been used for the sensitive and precise quantitation of both electrostatic and hydrophobic interactions of peptides and small molecules with lipid bilayers. We show that hydrophobic solutes are rapidly and quantitatively released from lipid dispersions when loaded onto a C4 reverse-phase HPLC column equilibrated in water+0.1% trifluoroacetic acid and that the lipid molecules have no interfering effect on the chromatography. Peptides interacting electrostatically with bilayers are released quantitatively when a higher ionic strength buffer (water+2% ammonium acetate) is used. As little as 50 ng of solute can be accurately quantitated even in the presence of milligram amounts of lipid. We demonstrate the application of these methods to the hydrophobic interactions between indoles and lipid bilayers and to the electrostatic interaction between defensins, which are cationic antibiotic peptides, and anionic bilayers. The high sensitivity allows nondestructive quantitation of submicrogram amounts of precious solutes and the high precision allows the heat capacity change, an important thermodynamic parameter, to be obtained from the partitioning data

Membrane partitioning: distinguishing bilayer effects from the hydrophobic effect.

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the concept of the "nonclassical" hydrophobic effect and the idea that the "classical" hydrophobic effect may not drive partitioning in many bilayer systems. We show through measurements of the heat capacity changes associated with the partitioning of tryptophan side-chain analogs into lipid bilayers and into bulk cyclohexane that the hydrophobic effect plays a crucial role regardless of the large negative enthalpy. The results emphasize that bulk-phase measurements are inadequate for describing bilayer partitioning. The experimental approach described should be generally useful for analyzing the bilayer interactions of a broad range of biologically important molecules

Partitioning of tryptophan side-chain analogs between water and cyclohexane.

We have measured the partitioning of the tryptophan side-chain analogs 3-methylindole and N-methylindole between water and cyclohexane over the temperature range 8-55 degrees C to investigate the relative contribution of the imine-NH- to the free energy of transfer. We take advantage of the fact that the indole imine nitrogen is blocked by a methyl group in N-methylindole. Unlike previous studies, we take into account the water present in the cyclohexane phase. Free energies of partitioning were calculated using mole-fraction, volume-fraction, and Flory-Huggins-corrected volume-fraction partition coefficients [De Young, L. R., & Dill, K. A. (1990) J. Phys. Chem. 94, 801-809; Sharp, K. A., Nicholls, A., Friedman, R., & Honig, B. (1991) Biochemistry 30, 9686-9697]. These approaches account for configurational entropy changes in different ways and thus lead to different values for the calculated free energies of transfer. There is a 2-3-fold difference in the free energies calculated from our measurements, using the different units. Independent of units, the partitioning of both compounds involves identical entropy changes. However, 3-methylindole has an additional unfavorable enthalpic contribution to partitioning into cyclohexane of +1.6 kcal/mol (independent of units) which is presumably the cost of removing the indole -NH- group from water and transferring it to cyclohexane. In cyclohexane, 3-methylindole forms hydrogen bonds with water that cause water to copartition into cyclohexane with the solute. A method is described which allows the partitioning process to be examined independent of subsequent interactions with water in the solvent

Evaluating Active U: an Internet-mediated physical activity program.

Background: Engaging in regular physical activity can be challenging, particularly during the winter months. To promote physical activity at the University of Michigan during the winter months, an eight-week Internet-mediated program (Active U) was developed providing participants with an online physical activity log, goal setting, motivational emails, and optional team participation and competition. Methods: This study is a program evaluation of Active U. Approximately 47,000 faculty, staff, and graduate students were invited to participate in the online Active U intervention in the winter of 2007. Participants were assigned a physical activity goal and were asked to record each physical activity episode into the activity log for eight weeks. Statistics for program reach, effectiveness, adoption, and implementation were calculated using the Re-Aim framework. Multilevel regression analyses were used to assess the decline in rates of data entry and goal attainment during the program, to assess the likelihood of joining a team by demographic characteristics, to test the association between various predictors and the number of weeks an individual met his or her goal, and to analyze server load. Results: Overall, 7,483 individuals registered with the Active U website (≈16% of eligible), and 79% participated in the program by logging valid data at least once. Staff members, older participants, and those with a BMI < 25 were more likely to meet their weekly physical activity goals, and average rate of meeting goals was higher among participants who joined a competitive team compared to those who participated individually (IRR = 1.28, P < .001). Conclusion: Internet-mediated physical activity interventions that focus on physical activity logging and goal setting while incorporating team competition may help a significant percentage of the target population maintain their physical activity during the winter months

Measurement of the antineutrino neutral-current elastic differential cross section

arXiv:1309.7257v1 [hep-ex