Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heatinduced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 908 light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 908 light scattering can be done simultaneously with far-or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein-lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.Keywords: circular dichroism spectroscopy; irreversible protein unfolding; turbidity; light scattering; asparaginase-2; high-density lipoprotein; amyloid; protein structure/folding; conformational changes; stability and mutagenesis; enzymes; thermodynamics; hydrodynamics; aggregation Thermal or chemical unfolding is generally used to determine thermodynamic protein stability, which is the Gibbs free energy difference between the folded and the unfolded states, DG = G U -G F . In thermal unfolding experiments, protein solution is heated at a constant rate, and changes in the protein conformation or their heat effects are monitored by spectroscopy or differential scanning calorimetry (DSC), respectively. The results, including the melting temperature (T m ), enthalpy (DH(T m )), and heat capacity increment (DC p ) of the unfolding, are used to determine protein stability function (DG(T)) (Privalov 1979;Pace et al. 1989). The key assumption behind this approach is that the protein unfolding is a thermodynamically reversible (that is, an equilibrium) transition. Although this assumption is usually valid for chemical denaturation, thermal denaturation (which is particularly widely used in protein stability studies) is often irreversible. The general root 3 These authors contributed equally to this work.

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.

Protein binding to phospholipid surface is commonly mediated by amphipathic α-helices. To understand the role of α-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoyl phosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kD) or its mutants containing single Pro substitutions along the sequence and differing in their α-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher α-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at T c =24 °C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g. at the lipid phase boundary), protein insertion into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion is aided by other surface-bound proteins and depends on their helical propensity: the larger the propensity the faster the binding and the broader its temperature range. This positive cooperativity in α-helical binding to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding. KeywordsHigh-density lipoprotein; kinetic stability; lipid fusion; vesicle clearance; atherosclerosis Protein binding to phospholipid surface is an important step in many biological reactions including apolipoprotein exchange among plasma lipoproteins, activation of lipid-regulated enzymes such as phosphoglycerate kinase (PGK) or CTP:phosphocholine cytidylyltransferase (CCT), synuclein aggregation in Parkinson's disease, lipid storage in adypocytes, and binding of antimicrobial peptides to cell surfaces. The binding depends on the structural and physicochemical properties of both proteins and lipids and is facilitated by the hydrophobic defects on the lipid surface that form primary protein binding sites (1-4). The common lipid surface-binding motif found in many proteins, including apolipoproteins, perilipin, synucleins, CTT and PGK, is amphipathic α-helix comprised of 11-mer sequence repeats ((5,6) and references therein). The extended apolar face of such a helix is optimized for interactions with Corresponding author: Dr. Olga Gursky, Department of Physiology and Biophysics, W329, Boston University School of Medicine, 715 Albany Street, Boston MA 02118, E-mail: gursky@bu.edu, Phone: (617) FAX: (617) NIH-PA Author ManuscriptNIH-PA Author Manuscri...

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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.