The excess heat capacity (Δ C ) of mixtures of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Chol) is examined in detail in large unilamellar vesicles (LUVs), both experimentally, using differential scanning calorimetry (DSC), and theoretically, using a three-state Ising model. The model postulates that DPPC can access three conformational states: gel, liquid-disordered (L), and liquid-ordered (L). The L state, however, is only available if coupled with interaction with an adjacent Chol. Δ C was calculated using Monte Carlo simulations on a lattice and compared to experiment. The DSC results in LUVs are compared with literature data on multilamellar vesicles (MLVs). The enthalpy change of the complete phase transition from gel to L is identical in LUVs and MLVs, and the melting temperatures ( T) are similar. However, the DSC curves in LUVs are significantly broader, and the maxima of Δ C are accordingly smaller. The parameters in the Ising model were chosen to match the DSC curves in LUVs and the nearest-neighbor recognition (NNR) data. The model reproduces the NNR data very well. It also reproduces the phase transition in DPPC, the freezing point depression induced by Chol, and the broad component of Δ C in DPPC/Chol LUVs. However, there is a sharp component, between 5 and 15 mol % Chol, that the model does not reproduce. The broad component of Δ C becomes dominant as Chol concentration increases, indicating that it involves melting of the L phase. Because the simulations reproduce this component, the conclusions regarding the nature of the phase transition at high Chol concentrations and the structure of the L phase are important: there is no true phase separation in DPPC/Chol LUVs. There are large domains of gel and L phase coexisting below T of DPPC, but above T the three states of DPPC are mixed with Chol, although clusters persist.
The excess heat capacity functions (ΔCp) associated with the main phase transition of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) are very different. Two explanations are possible. First, the difference in vesicle size (curvature) results in different gel-fluid interactions in the membrane; those interactions have a large effect on the cooperativity of the phase transition. Second, there is communication between the bilayers in an MLV when they undergo the gel-fluid transition; this communication results in thermodynamic coupling of the phase transitions of the bilayers in the MLV and, consequently, in an apparent increase in the cooperativity of the transition. To test these hypotheses, differential scanning calorimetry was performed on giant unilamellar vesicles (GUVs) of pure dipalmitoylphosphatidylcholine. The ΔCp curve of GUVs was found to resemble that of the much smaller LUVs. The transition in GUVs and LUVs is much broader (half-width ∼1.5°C) than in MLVs (∼0.1°C). This similarity in GUVs and LUVs indicates that their size has little effect on gel-fluid interactions in the phase transition. The result suggests that coupling between the transitions in the bilayers of an MLV is responsible for their apparent higher cooperativity in melting.
One of the long-standing issues surrounding cholesterol (Chol) relates to its two-faced character. In particular, the consequences of its having a rough β-face and a smooth α-face on its structural influence in cell membranes has remained elusive. In this study, direct comparisons have been made between cholesterol and a “smoothened” analog, DChol (i.e., 18,19-dinorcholesterol) using model membranes and a combination of nearest-neighbor recognition, differential scanning calorimetry, fluorescence, and monolayer measurements. Taken together, these results indicate that subtle differences exist between the interaction of these two sterols with the different states of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Chol has a greater condensing power than DChol, but only slightly so, i.e., on the order of a few tens of calories per mole.
The spring region of the giant muscle protein titin contains many immunoglobulin-like (Ig) domains, which unfold and refold under low (physiological) stretch forces. Small heat shock proteins such as alphaB-crystallin (aBC) translocate under physiological or pathological stress to the titin springs. To better understand this protective function we studied the unfolding-refolding behavior of an 8-Ig-domain titin construct (I91) 8 by single-molecule AFM force spectroscopy, in the absence/presence of recombinant aBC (pH7; pH6; or pH5) or 'control' protein of similar size. Titin Ig domains were unfolded at 175 pN constant force applied for a variable ''denature'' time (t D ), then the force was set to zero for a variable ''quench'' time (t Q ) to allow for domain refolding, and finally a ''probe'' pulse (175 pN; t P = 5 s) was applied to test how many domains had refolded. Interestingly, Ig domain unfolding kinetics were little affected by aBC. However, upon lowering pH from 7 to 6, the refolded fraction (number of refolded Ig domains during t Q indexed to number of unfolded Ig domains during t D ) decreased slightly, indicating domain misfolding. At pH5, the refolded fraction dropped by half and~50% of titin Ig domains showed misfolding events, an effect independent of t D (variation, 2 -40 s). Importantly, aBC (10 mM or 20 mM) normalized the refolded fraction to values observed at pH7, whereas control protein had no such protective effect. The refolded fraction depended strongly on t Q (variation, 0.5 -10 s), under all experimental conditions. Ig domain refolding kinetics were greatly slowed by lowering pH from 7 to 5, as quantified on refolded fraction vs. t Q plots, on which means were fit by simple exponentials. Again, aBC normalized the refolding kinetics to those observed at pH7 in the absence of aBC, whereas control protein had no such effect. We conclude that aBC speeds up titin Ig domain refolding (novel foldase activity!) and protects from domain misfolding, especially under acidic stress, which is frequently encountered in muscle cells.
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