The enthalpy of transfer of four different amphiphilic molecules from the aqueous phase to the lipid membrane was determined by titration calorimetry. The four molecules investigated were the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), the membrane conductivity inducing anion tetraphenylborate (TPB), the Ca2+ channel blocker amlodipine [Bäuerle, H. D., & Seelig, J. (1991) Biochemistry 30, 7203-7211], and the positively charged local anesthetic dibucaine. All four amphiphiles penetrate into the hydrophobic part of the membrane, and their binding constants, after correcting for electrostatic effects, range between 600 M-1 for dibucaine and 60,000 M-1 for tetraphenylborate. The corresponding changes in free energy were about -6 to -9 kcal/mol. Binding of the amphiphiles to membrane vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was accompanied by exothermic heats of reaction for all four molecules. For TNS, TPB, and amlodipine, the enthalpies of transfer were almost identical and corresponded to delta H approximately -9 kcal/mol, essentially accounting for the total free energy change. Thus, the binding of these charged amphiphiles to the hydrophobic membrane was driven by enthalpy. This is in contrast to the classical hydrophobic effect, where the transfer is considered to be entropy driven. For dibucaine, the enthalpy of transfer was smaller with delta H approximately -2 kcal/mol but was still about one-third of the total free energy change. All enthalpies of transfer exhibited a distinct temperature dependence with molar heat capacities delta Cp of -30 to -100 cal mol-1K-1 for the transfer from water to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclodextrins are able to bind hydrophobic molecules in their interior cavity and as such have received a great deal of attention as carriers of cholesterol, lipophilic drugs, and other sparingly soluble compounds. Despite the importance of these biochemical applications, relatively little is known about the interactions of cyclodextrins with phospholipid membranes. Here we characterize the binding of randomly methylated β-cyclodextrin (mβCD) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using right-angle light scattering and isothermal titration calorimetry. Existing models of lipophile−membrane interactions are inadequate to describe the observed binding; we introduce a modified chemical reaction model in which the chemical activity of the phospholipid is independent of its concentration. We find that an average of four mβCD molecules bind to each POPC molecule with an enthalpy of reaction of 46 kJ mol-1 and an equilibrium constant of 90 M-3. These results are consistent with earlier qualititative observations and suggest that disruption of phospholipid membranes may be minimized if the concentration of mβCD is kept below about 15 mM.

The interaction of four structurally related somatostatin analogues (effective electric charge +0.4 < or = < or = +3) with lipid membranes was studied with titration calorimetry and was compared with the functional activity of the peptides. Surface activity measurements provided average cross-sections of 70 or 135 A2, indicating that the cyclic molecules orient at the air-water interface with their ring system either parallel (z = +3) or perpendicular (z = +1) to the surface or switching between the two orientations according to the surface density (z = +2). The nonspecific binding of the peptides to sonified lipid vesicles was enthalpy-driven with a delta H of -4 to -7.5 kcal/mol. A consistent quantitative analysis of the binding isotherms was achieved by combining electrostatic attractions, calculated via the Gouy-Chapman theory, with a nonspecific surface partition equilibrium for the nonpolar interactions. The electrostatic attraction of the cationic peptides varied strongly according to the peptide charge. Due to the flat ring structure of the cyclic peptides, their true physical charge was sensed at the membrane surface, and no "charge screening" was observed. Peptide binding to the negative charged membrane was accompanied by a proton-uptake of the N-terminal amino group of 0.23-0.38 H+/peptide. Deviations from the theoretical prediction of 0.39 H+/peptide can be explained by a preferential binding of the nonprotonated species. The nonpolar interactions, as described by the surface partition coefficients of the four peptides, fell into a narrow range of K congruent to 50-230 M-1 whereas the apparent overall binding constants were between 200 and 5000 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Hsp90, an abundant heat shock protein that is highly expressed even under physiological conditions, is involved in the folding of key molecules of the cellular signal transduction system such as kinases and steroid receptors. It seems to contain two chaperone sites differing in substrate specificity. Binding of ATP or the antitumor drug geldanamycin alters the substrate affinity of the N-terminal chaperone site, whereas both substances show no inf luence on the C-terminal one. In wild-type Hsp90 the fragments containing the chaperone sites are connected by a highly charged linker of various lengths in different organisms. As this linker region represents the most striking difference between bacterial and eukaryotic Hsp90s, it may be involved in a gain of function of eukaryotic Hsp90s. Here, we have analyzed a fragment of yeast Hsp90 consisting of the N-terminal domain and the charged region (N272) in comparison with the isolated N-terminal domain (N210). We show that the charged region causes an increase in the affinity of the N-terminal domain for nonnative protein and establishes a crosstalk between peptide and ATP binding. Thus, the binding of peptide to N272 decreases its affinity for ATP and geldanamycin, whereas the ATP-binding properties of the monomeric N-terminal domain N210 are not inf luenced by peptide binding. We propose that the charged region connecting the two chaperone domains plays an important role in regulating chaperone function of Hsp90.