Biomedical subjects

Donald L Gantz

Publications and source records attributed to Donald L Gantz.

8 recordsLinked to original sources

PubMed2006

Interactions between fatty acids and alpha-synuclein.

alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether alphaS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of alphaS signal in both (1)H and (13)C spectra and ii) the appearance of a very broad FA (13)C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific alphaS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of alphaS, indicating a direct protein-lipid interaction. We conclude that alphaS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of alphaS that is most likely related to its physiological role(s) in the cell.

Animals↗

PubMed2006

Effects of salt on the thermal stability of human plasma high-density lipoprotein.

High-density lipoproteins (HDL) mediate cholesterol removal and thereby protect against atherosclerosis. Mature spherical HDL contain the apolar lipid core and polar surface of proteins and phospholipids. Earlier, we showed that the structural integrity of HDL is modulated by kinetic barriers that prevent spontaneous protein dissociation and lipoprotein fusion and rupture. To determine the role of electrostatic interactions in the kinetic stability of mature HDL, here we analyze the effects of salt and pH on their thermal denaturation. In low-salt buffer at pH 5.7-7.7, HDL are highly thermostable. Increasing the salt concentration from 0 to 0.3 M NaCl causes low-temperature shifts in the calorimetric HDL transitions of up to -14 degrees C. This salt-induced destabilization leads to protein unfolding below 100 degrees C, facilitating the first Arrhenius analysis of HDL denaturation by circular dichroism spectroscopy. In 150 mM NaCl, two kinetic phases in HDL protein unfolding are observed: a faster phase with an activation energy E(a,fast) < or =15 kcal/mol and a slower phase with an E(a,slow) = 50 +/- 7 kcal/mol. Gel electrophoresis and electron microscopic data suggest that the faster phase involves partial protein unfolding but no significant protein dissociation or changes in HDL size, while the slower phase involves complete protein unfolding, partial protein dissociation, and HDL fusion. Hence, the slower phase may resemble HDL remodeling and fusion by plasma enzymes during metabolism. Analysis of the effects of various salts, sucrose, and pH suggests that HDL destabilization by salt results from ionic screening of favorable short-range electrostatic interactions such as salt bridges. Consequently, electrostatic interactions significantly contribute to the high thermostability of HDL in low-salt solutions.

Calorimetry, Differential Scanning↗

PubMed2005

Kinetic stabilization and fusion of apolipoprotein A-2:DMPC disks: comparison with apoA-1 and apoC-1.

Denaturation studies of high-density lipoproteins (HDL) containing human apolipoprotein A-2 (apoA-2) and dimyristoyl phosphatidylcholine indicate kinetic stabilization. Circular dichroism (CD) and light-scattering melting curves show hysteresis and scan rate dependence, indicating thermodynamically irreversible transition with high activation energy E(a). CD and light-scattering data suggest that protein unfolding triggers HDL fusion. Electron microscopy, gel electrophoresis, and differential scanning calorimetry show that such fusion involves lipid vesicle formation and dissociation of monomolecular lipid-poor protein. Arrhenius analysis reveals two kinetic phases, a slower phase with E(a,slow) = 60 kcal/mol and a faster phase with E(a,fast) = 22 kcal/mol. Only the fast phase is observed upon repetitive heating, suggesting that lipid-poor protein and protein-containing vesicles have lower kinetic stability than the disks. Comparison of the unfolding rates and the melting data recorded by differential scanning calorimetry, CD, and light scattering indicates the rank order for the kinetic disk stability, apoA-1 > apoA-2 > apoC-1, that correlates with protein size rather than hydrophobicity. This contrasts with the tighter association of apoA-2 than apoA-1 with mature HDL, suggesting different molecular determinants for stabilization of model discoidal and plasma spherical HDL. Different effects of apoA-2 and apoA-1 on HDL fusion and stability may reflect different metabolic properties of apoA-2 and/or apoA-1-containing HDL.

Apolipoprotein A-I↗

PubMed2004

Poly(ethylene glycol)-induced fusion and destabilization of human plasma high-density lipoproteins.

High-density lipoproteins (HDL) are macromolecular complexes of specific proteins and lipids that mediate the removal of cholesterol from peripheral tissues. Chemical unfolding revealed that HDL fusion and rupture are the two main kinetic steps in HDL denaturation. Here we test the hypothesis that lipid fusogens such as poly(ethylene glycol) (PEG) may promote lipoprotein fusion and rupture and thereby destabilize HDL. We analyze thermal disruption of spherical HDL in 0-15% PEG-8000 by calorimetric, spectroscopic, electron microscopic, and light scattering techniques. We demonstrate that the two irreversible high-temperature endothermic HDL transitions involve particle enlargement and show a heating rate dependence characteristic of kinetically controlled reactions with high activation energy. The first calorimetric transition reflects HDL fusion and dissociation of lipid-poor apolipoprotein A-1 (apoA-1), and the second transition reflects HDL rupture and release of the apolar lipid core. Neither transition involves substantial protein unfolding; thus, the transition heat originates from lipid and/or protein dissociation and repacking. At room temperature, PEG-8000 induces HDL fusion that is distinct from the heat-, denaturant-, or enzyme-induced fusion since it leads to formation of larger particles and does not involve apoA-1 dissociation. Increasing the PEG concentration in solution from 0 to 15% leads to low-temperature shifts by approximately -18 degrees C in the two calorimetric HDL transitions without altering their nature. Thus, consistent with our hypothesis, PEG-8000 induces fusion and reduces the thermal stability of HDL. Our results suggest that PEG is useful for the analysis of the molecular events involved in metabolic HDL remodeling and fusion.

Apolipoprotein A-I↗

PubMed2003

Nickel (Ni2+) enhancement of microtubule assembly in vitro is dependent on GTP function.

Microtubule (MT) assembly in vitro is accompanied by hydrolysis of tubulin-bound GTP at E-site. Ni2+, a human carcinogen, has been shown to markedly perturb the MT system in cultured cells and enhance MT assembly in vitro. To further probe the mechanisms of such multiple Ni2+ damaging actions on MT, we have focused on dissecting the role of the Ni2+/GTP interaction in influencing MT assembly in vitro as monitored by a turbidity assay at A350 at 27 degrees C using purified bovine brain MT proteins containing 162 microM each of Mg2+ and EGTA. MT assembly was initiated by addition of GTP and progressed in a GTP dose-dependent manner. The minimal and optimal exogenous [GTP] required for MT assembly were 15.6 and 500 microM, respectively. Replacement of GTP (25-87%) with increasing [NiCl2] while keeping the sum of [GTP] and [Ni2+] constant at 500 microM enabled MT assembly to proceed with shortened "lags" but reaching the same maximum plateau levels or elongation rates as with 500 microM GTP only. However, in reactions with Ni2+ replacing >94% of GTP, marked inhibition of MT assembly (lower plateaus) occurred. Electron microscopic (EM) examinations showed that MT formed with high Ni2+ substitutions for GTP appeared shorter, more numerous, and resistant to Ca2+ disruption than those assembled with 500 microM GTP only. Notably, in the presence of 500 microM Ni2+ with no GTP added, no typical MT were observed under EM, despite increases in turbidity of the reaction. In addition, the critical concentration of MT proteins required for assembly was also considerably decreased under conditions of Ni2+ replacements of GTP. These results point to an important role of GTP/Ni2+ interaction in modulating the Ni2+ enhancement of MT assembly in vitro.

Animals↗

PubMed2003

Effects of mutations in apolipoprotein C-1 on the reconstitution and kinetic stability of discoidal lipoproteins.

To probe the role of protein conformation in the formation and kinetic stability of discoidal lipoproteins, thermal unfolding and refolding studies were carried out using model lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and selected mutants of human apolipoprotein C-1 (apoC-1). Circular dichroism (CD) spectroscopy and electron microscopy show that the Q31P mutant, which has alpha-helical content in solution (33%) and on DMPC disks (67%) similar to that of the wild type (WT), forms disks of smaller diameter, = 13 nm, compared to 17 nm of the WT-DMPC disks. The L34P mutant, which is largely unfolded in solution, forms disks with alpha-helix content and diameter similar to those of the WT. The R23P mutant, which is fully unfolded in solution, forms disks that have similar diameter but reduced alpha-helix content (40%) compared to the WT-DMPC disks (65%). Remarkably, despite large variations in the alpha-helix content or the disk diameter among different mutant-DMPC complexes, the mutations have no significant effect on the unfolding rates or the Arrhenius activation energy of the disk denaturation, E(a) = 25-29 kcal/mol. This suggests that the kinetic stability of the discoidal complexes is dominated by the lipid-lipid rather than the protein-lipid interactions. In contrast to the heat denaturation, the lipoprotein reconstitution upon cooling monitored by CD and light scattering is significantly affected by mutations, with Q31P forming disks in the broadest and R23P in the narrowest temperature range. Our results suggest that the apolipoprotein helical structure in solution facilitates reconstitution of discoidal lipoproteins but has no significant effect on their kinetic stability.

Apolipoprotein C-I↗

PubMed2003

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.

Circular Dichroism↗

PubMed2002

Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?

Thermal unfolding of discoidal complexes of apolipoprotein (apo) C-1 with dimyristoyl phosphatidylcholine (DMPC) reveals a novel mechanism of lipoprotein stabilization that is based on kinetics rather than thermodynamics. Far-UV CD melting curves recorded at several heating/cooling rates from 0.047 to 1.34 K/min show hysteresis and scan rate dependence characteristic of slow nonequilibrium transitions. At slow heating rates, the apoC-1 unfolding in the complexes starts just above 25 degrees C and has an apparent melting temperature T(m) approximately 48 +/- 1.5 degrees C, close to T(m) = 51 +/- 1.5 degrees C of free protein. Thus, DMPC binding may not substantially increase the low apparent thermodynamic stability of apoC-1, DeltaG(25 degrees C) < 2 kcal/mol. The scan rate dependence of T(m) and Arrhenius analysis of the kinetic data suggest an activation enthalpy E(a) = 25 +/- 5 kcal/mol that provides the major contribution to the free energy barrier for the protein unfolding on the disk, DeltaG > or = 17 kcal/mol. Consequently, apoC-1/DMPC disks are kinetically but not thermodynamically stable. To explore the origins of this kinetic stability, we utilized dynode voltage measured in CD experiments that shows temperature-dependent contribution from UV light scattering of apoC-1/DMPC complexes (d approximately 20 nm). Correlation of CD and dynode voltage melting curves recorded at 222 nm indicates close coupling between protein unfolding and an increase in the complex size and/or lamellar structure, suggesting that the enthalpic barrier arises from transient disruption of lipid packing interactions upon disk-to-vesicle fusion. We hypothesize that a kinetic mechanism may provide a general strategy for lipoprotein stabilization that facilitates complex stability and compositional variability in the absence of high packing specificity.

Apolipoprotein C-I↗