Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

Abstract

High-density lipoproteins (HDL) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α-helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule-like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA-I, shows relatively large volume contraction, ΔV unf =-0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα unf ≤0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α-helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA-I shows large thermal expansion coefficient, α(5°) = 15·10 -4 K -1 , that rapidly declines upon heating from 5-40°C, α(40°)-α(5°)=-4·10 -4 K -1 ; apolipoprotein C-I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein-lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA-I or apoC-I, dimyristoyl phosphatidylcholine, and 0-20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport.

Pressure Perturbation Calorimetry of Lipoproteins Reveals an Endothermic Transition without Detectable Volume Changes. Implications for Adsorption of Apolipoprotein to a Phospholipid Surface

“…This is consistent with the absence of spontaneous remodeling of DPPC MLV by apoA-I. The broad peak centered at T m = 60 °C (Figure 6B) reflects unfolding of lipid-free apoA-I (ref 27 and references therein). Therefore, the apoA-I unfolding assessed by CD and DSC is not affected by the presence of DPPC MLV.…”

“…Because of instrumental limitations of VP-DSC, such data could be recorded only by using a lipid:protein molar ratio of no more than 40:1 while maintaining a protein concentration of at least 0.5 mg/mL that is necessary to monitor apoA-I unfolding by PPC. 27 This lipid:protein ratio is close to that found in rHDL; hence, sufficient lipid was present in the samples to significantly affect apolipoprotein hydration. Nevertheless, the calorimetric data of apoA-I that was free in solution 27 or in the presence of SUV were similar and showed endothermic protein unfolding at T m = 60 °C observed by DSC, accompanied by a negative volume change observed by PPC (Figure 8A,B).…”

“…27 We also conducted PPC analysis of lipid-free apoA-I in solution over a broad temperature range that encompasses protein unfolding centered at T m = 60 °C. The results demonstrated that apoA-I unfolding involves a relatively large negative volume change (Δ V unf = −0.33%) and showed the importance of charged groups for apolipoprotein hydration.…”

“…The results demonstrated that apoA-I unfolding involves a relatively large negative volume change (Δ V unf = −0.33%) and showed the importance of charged groups for apolipoprotein hydration. 27 In this work, we analyze a variety of model and plasma lipoproteins by PPC over a temperature range that encompasses lipoprotein heat denaturation.…”

Impact of Self-association on Function of Apolipoprotein A-I

“…S3), and other globular proteins (64). In agreement with the DSC results, these unusually large volume changes in ⌬W[High] may be due to the combined effect of secondary structure unfolding and self-association disruption, which contribute to increasing the solvent exposure of charged groups otherwise restricted in a solvent-excluded environment within self-associated species.…”