Olga Gursky

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments

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, heat‐induced 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 90° light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90° 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.

Temperature-dependent β-sheet formation in β-amyloid Aβ1–40 peptide in water: uncoupling β-structure folding from aggregation

To probe the role of temperature in the conversion of soluble Alzheimers beta-amyloid peptide (Abeta) to insoluble beta-sheet rich aggregates, we analyzed the solution conformation of Abeta(1-40) from 0 to 98 degrees C by far-UV circular dichroism (CD) and native gel electrophoresis. The CD spectra of 15-300 microg/ml Abeta(1-40) in aqueous solution (pH approximately 4.6) at 0 degrees C are concentration-independent and suggest a substantially unfolded and/or unusually folded conformation characteristic of Abeta monomer or dimer. Heating from 0 to 37 degrees C induces a rapid reversible coil to beta-strand transition that is independent of the peptide concentration and thus is not linked to oligomerization. Consequently, this transition may occur within the Abeta(1-40) monomer or dimer. Incubation at 37 degrees C leads to slow reversible concentration-dependent beta-sheet accumulation; heating to 85 degrees C induces further beta-sheet folding and oligomerization. Our results demonstrate the importance of temperature and thermal history for the conformation of Abeta.

Human Plasma High-density Lipoproteins are Stabilized by Kinetic Factors

High-density lipoproteins (HDL) are heterogeneous complexes of proteins and lipids that mediate cholesterol removal from the body. Our thermal and chemical denaturation studies of mature spherical HDL isolated from human plasma show that, contrary to the widely held assumption, the particle stability has a kinetic rather than thermodynamic origin. Guanidinum hydrochloride (GdmHCl) concentration jumps at 25 degrees C monitored by circular dichroism (CD) at 222 nm reveal two dominant irreversible kinetic phases in HDL denaturation. The slower phase (relaxation time tau(1) approximately 2 x 10(4) seconds) is observed in 1-6 M GdmHCl, and the faster phase (tau(2) approximately 2 x 10(3) seconds) is detected in 3-6 M GdmHCl. Comparison of the free energy barriers associated with these phases, deltaG* = 16-17 kcal mol(-1), with the near-zero apparent thermodynamic stability inferred from the spectroscopic measurements after prolonged incubation in 0-6 M GdmHCl at 22 degrees C indicates the kinetic origin for HDL stabilization. Electron microscopic analysis of HDL incubated in 0-6 M GdmHCl suggests that the slower kinetic phase involves HDL fusion, while the faster phase involves particle rupture and release of the apolar lipid core. Thermal denaturation experiments indicate high enthalpic barriers for the particle rupture that may arise from the transient disruption of lipid and/or protein packing interactions. These results corroborate our earlier analysis of model discoidal HDL and indicate that a kinetic mechanism provides a universal natural strategy for lipoprotein stabilization. Such a mechanism may facilitate structural integrity of the heterogeneous lipoprotein particles, slow their spontaneous interconversions, and thereby modulate lipoprotein lifetime and functions.

The crystal structure of the C-terminal truncated apolipoprotein A-I sheds new light on amyloid formation by the N-terminal fragment.

Apolipoprotein A-I (apoA-I) is the main protein of plasma high-density lipoproteins (HDL, or good cholesterol) that remove excess cell cholesterol and protect against atherosclerosis. In hereditary amyloidosis, mutations in apoA-I promote its proteolysis and the deposition of the 9-11 kDa N-terminal fragments as fibrils in vital organs such as kidney, liver, and heart, causing organ damage. All known amyloidogenic mutations in human apoA-I are clustered in two residue segments, 26-107 and 154-178. The X-ray crystal structure of the C-terminal truncated human protein, Δ(185-243)apoA-I, determined to 2.2 Å resolution by Mei and Atkinson, provides the structural basis for understanding apoA-I destabilization in amyloidosis. The sites of amyloidogenic mutations correspond to key positions within the largely helical four-segment bundle comprised of residues 1-120 and 144-184. Mutations in these positions disrupt the bundle structure and destabilize lipid-free apoA-I, thereby promoting its proteolysis. Moreover, many mutations place a hydrophilic or Pro group in the middle of the hydrophobic lipid-binding face of the amphipathic α-helices, which will likely shift the population distribution from HDL-bound to lipid-poor/free apoA-I that is relatively unstable and labile to proteolysis. Notably, the crystal structure shows segment L44-S55 in an extended conformation consistent with the β-strand-like geometry. Exposure of this segment upon destabilization of the four-segment bundle probably initiates the α-helix to β-sheet conversion in amyloidosis. In summary, we propose that the amyloidogenic mutations promote apoA-I proteolysis by destabilizing the protein structure not only in the lipid-free but also in the HDL-bound form, with segment L44-S55 providing a likely template for the cross-β-sheet conformation.

Conformational changes in cubic insulin crystals in the pH range 7–11

To determine the effect of variations in the charge distribution on the conformation of a protein molecule, we have solved the structures of bovine cubic insulin over a pH range from 7 to 11 in 0.1 M and 1 M sodium salt solutions. The x-ray data were collected beyond 2-A resolution and the R factors for the refined models ranged from 0.16 to 0.20. Whereas the positions of most protein and well-ordered solvent atoms are conserved, about 30% of residues alter their predominant conformation as the pH is changed. Conformational switching of A5 Gln and B10 His correlates with the pH dependence of monovalent cation binding to insulin in cubic crystals. Shifts in the relative positions of the A chain NH2-terminal and B chain COOH-terminal groups are probably due to titration of the A1 alpha-amino group. Two alternative positions of B25 Phe and A21 Asn observed in cubic insulin at pH 11 are similar to those found in two independent molecules of the 2Zn insulin dimer at pH 6.4. The conformational changes of the insulin amino acids appear to be only loosely coupled at distant protein sites. Shifts in the equilibrium between distinct conformational substates as the charge distribution on the protein is altered are analogous to the electrostatically triggered movements that occur in many functional protein reactions.

The Critical Role of the Constant Region in Thermal Stability and Aggregation of Amyloidogenic Immunoglobulin Light Chain

Light chain (LC) amyloidosis (AL) is a fatal disease in which immunoglobulin LC deposit as fibrils. Although the LC amyloid-forming propensity is attributed primarily to the variable region, fibrils also contain full-length LC comprised of variable-joining (V(L)) and constant (C(L)) regions. To assess the role of C(L) in fibrillogenesis, we compared the thermal stability of full-length LC and corresponding V(L) and C(L) fragments. Protein unfolding and aggregation were monitored by circular dichroism and light scattering. A full-length λ6 LC purified from urine of a patient with AL amyloidosis showed irreversible unfolding coupled to aggregation. The transition temperature decreased at slower heating rates, indicating kinetic effects. Next, we studied five recombinant λ6 proteins: full-length amyloidogenic LC, its V(L), germline LC, germline V(L), and C(L). Amyloidogenic and germline proteins showed similar rank order of stability, V(L) < LC < C(L); hence, in the full-length LC, V(L) destabilizes C(L). Amyloidogenic proteins were less stable than their germline counterparts, suggesting that reduction in V(L) stability destabilizes the full-length LC. Thermal unfolding of the full-length amyloidogenic and germline LC required high activation energy and involved irreversible aggregation, yet the unfolding of the isolated V(L) and C(L) fragments was partially reversible. Therefore, compared to their fragments, full-length LCs are more likely to initiate aggregation during unfolding and provide a template for the V(L) deposition. The kinetic barrier for this aggregation is regulated by the stability of the V(L) region. This represents a paradigm shift in AL fibrillogenesis and suggests C(L) region as a potential therapeutic target.

Correlation of Structural Stability with Functional Remodeling of High-Density Lipoproteins: The Importance of Being Disordered†

High-density lipoproteins (HDLs) are protein-lipid assemblies that remove excess cell cholesterol and prevent atherosclerosis. HDLs are stabilized by kinetic barriers that decelerate protein dissociation and lipoprotein fusion. We propose that similar barriers modulate metabolic remodeling of plasma HDLs; hence, changes in particle composition that destabilize HDLs and accelerate their denaturation may accelerate their metabolic remodeling. To test this notion, we correlate existing reports on HDL-mediated cell cholesterol efflux and esterification, which are obligatory early steps in cholesterol removal, with our kinetic studies of HDL stability. The results support our hypothesis and show that factors accelerating cholesterol efflux and esterification in model discoidal lipoproteins (including reduced protein size, reduced fatty acyl chain length, and/or increased level of cis unsaturation) destabilize lipoproteins and accelerate their fusion and apolipoprotein dissociation. Oxidation studies of plasma spherical HDLs show a similar trend: mild oxidation by Cu(2+) or OCl(-) accelerates cell cholesterol efflux, protein dissociation, and HDL fusion, while extensive oxidation inhibits these reactions. Consequently, moderate destabilization may be beneficial for HDL functions by facilitating insertion of cholesterol and lipophilic enzymes, promoting dissociation of lipid-poor apolipoproteins, which are primary acceptors of cell cholesterol, and thereby accelerating HDL metabolism. Therefore, HDL stability must be delicately balanced to maintain the structural integrity of the lipoprotein assembly and ensure structural specificity necessary for interactions of HDL with its metabolic partners, while facilitating rapid HDL remodeling and turnover at key junctures of cholesterol transport. The inverse correlation between HDL stability and remodeling illustrates the functional importance of structural disorder in macromolecular assemblies stabilized by kinetic barriers.

Monovalent cation binding to cubic insulin crystals

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.

Apolipoprotein structure and dynamics

Purpose of review This review highlights recent advances in structural studies of exchangeable human apolipoproteins and the insights they provide into lipoprotein action in cardiovascular and amyloid diseases. Recent findings The high-resolution X-ray crystal structure of free apoA-II reveals a parallel helical array that may represent other lipid-poor apolipoproteins, and the structure in complex with detergent substantiates the belt model for the protein arrangement on lipoproteins. Nuclear magnetic resonance structures of apolipoprotein–detergent complexes show a repertoire of curved helical conformations, suggesting multiple helical arrangements on the lipid. Low-resolution spectroscopic analyses, interface studies and molecular modeling provide new insights into the ‘hinge-domain’ mechanism of apolipoprotein adaptation at variable lipoprotein surfaces. A kinetic mechanism for lipoprotein stabilization is proposed. Summary Cumulative evidence supports the belt model that provides a general structural basis for understanding the molecular mechanisms of functional apolipoprotein reactions, such as binding to lipoprotein receptors, lipid transporters, and the activation of lipophilic enzymes. However, the detailed protein and lipid conformations on lipoproteins and the underlying molecular interactions are unclear. New insights will hopefully emerge once the first detailed lipoprotein structure is solved.

Amyloidogenic mutations in human apolipoprotein A-I are not necessarily destabilizing - a common mechanism of apolipoprotein A-I misfolding in familial amyloidosis and atherosclerosis.

High‐density lipoproteins and their major protein, apolipoprotein A‐I (apoA‐I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, nonvariant full‐length apoA‐I deposits as fibrils in atherosclerotic plaques; in familial amyloidosis, N‐terminal fragments of variant apoA‐I deposit in vital organs, damaging them. Recently, we used the crystal structure of Δ(185–243)apoA‐I to show that amyloidogenic mutations destabilize apoA‐I and increase solvent exposure of the extended strand 44–55 that initiates β‐aggregation. In the present study, we test this hypothesis by exploring naturally occurring human amyloidogenic mutations, W50R and G26R, within or close to this strand. The mutations caused small changes in the proteins α‐helical content, stability, proteolytic pattern and protein–lipid interactions. These changes alone were unlikely to account for amyloidosis, suggesting the importance of other factors. Sequence analysis predicted several amyloid‐prone segments that can initiate apoA‐I misfolding. Aggregation studies using N‐terminal fragments verified this prediction experimentally. Three predicted N‐terminal amyloid‐prone segments, mapped on the crystal structure, formed an α‐helical cluster. Structural analysis indicates that amyloidogenic mutations or Met86 oxidation perturb native packing in this cluster. Taken together, the results suggest that structural perturbations in the amyloid‐prone segments trigger α‐helix to β‐sheet conversion in the N‐terminal ~ 75 residues forming the amyloid core. Polypeptide outside this core can be proteolysed to form 9–11 kDa N‐terminal fragments found in familial amyloidosis. Our results imply that apoA‐I misfolding in familial and acquired amyloidosis follows a similar mechanism that does not require significant structural destabilization or proteolysis. This novel mechanism suggests potential therapeutic interventions for apoA‐I amyloidosis.