Vasanthy Narayanaswami

The Lipid-associated Conformation of the Low Density Lipoprotein Receptor Binding Domain of Human Apolipoprotein E

Apolipoprotein E (apoE) is a 34-kDa exchangeable apolipoprotein that regulates metabolism of plasma lipoproteins by functioning as a ligand for members of the LDL receptor family. The receptor-binding region localizes to the vicinity of residues 130–150 within its independently folded 22-kDa N-terminal domain. In the absence of lipid, this domain exists as a receptor-inactive, globular four-helix bundle. Receptor recognition properties of this domain are manifest upon lipid association, which is accompanied by a conformational change in the protein. Fluorescence resonance energy transfer has been used to monitor helix repositioning, which accompanies lipid association of the apoE N-terminal domain. Site-directed mutagenesis was used to replace naturally occurring Trp residues with phenylalanine, creating a Trp-null apoE3 N-terminal domain (residues 1–183). Subsequently, tyrosine residues in helix 2, helix 3, or helix 4 were converted to Trp, generating single Trp mutant proteins. The lone cysteine at position 112 was covalently modified withN-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine, which serves as an energy acceptor from excited tryptophan residues. Fluorescence resonance energy transfer analysis of apoE N-terminal domain variants in phospholipid disc complexes suggests that the helix bundle opens to adopt a partially extended conformation. A model is presented that depicts a tandem arrangement of the receptor-binding region of the protein in the disc complex, corresponding to its low density lipoprotein receptor-active conformation.

Interaction of an Exchangeable Apolipoprotein with Phospholipid Vesicles and Lipoprotein Particles ROLE OF LEUCINES 32, 34, AND 95 IN LOCUSTA MIGRATORIAAPOLIPOPHORIN III

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipid surfaces. In the lipid-free state this 164-residue protein exists as a bundle of five elongated amphipathic α-helices. Upon lipid binding, apoLp-III undergoes a significant conformational change, resulting in exposure of its hydrophobic interior to the lipid environment. On the basis of x-ray crystallographic data (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603–608), it was proposed that hydrophobic residues, present in loops that connect helices 1 and 2 (Leu-32 and Leu-34) and helices 3 and 4 (Leu-95), may function in initiation of lipid binding. To examine this hypothesis, mutant apoLp-IIIs were designed wherein the three Leu residues were replaced by Arg, individually or together. Circular dichroism spectroscopy and temperature and guanidine hydrochloride denaturation studies showed that the mutations did not cause major changes in secondary structure content or stability. In lipid binding assays, addition of apoLp-III to phospholipid vesicles caused a rapid clearance of vesicle turbidity due to transformation to discoidal complexes. L34R and L32R/L34R/L95R apoLp-IIIs displayed a much stronger interaction with lipid vesicles than wild-type apoLp-III. Furthermore, it was demonstrated that the mutant apoLp-IIIs retained their ability to bind to lipoprotein particles. However, in lipoprotein competition binding assays, the mutants displayed an impaired ability to initiate a binding interaction when compared with wild-type apoLp-III. The data indicate that the loops connecting helices 1 and 2 and helices 3 and 4 are critical regions in the protein, contributing to recognition of hydrophobic defects on lipoprotein surfaces by apoLp-III.

Fluorescence Studies of Exchangeable Apolipoprotein-Lipid Interactions SUPERFICIAL ASSOCIATION OF APOLIPOPHORIN III WITH LIPOPROTEIN SURFACES

Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an 18-kDa exchangeable apolipoprotein that reversibly associates with lipoprotein particles. In the absence of lipid, apoLp-III exists as an elongated bundle of five amphipathic α-helices. Upon lipid association, the protein is postulated to undergo a major conformational change, wherein the bundle opens around hinge loop regions, resulting in exposure of its hydrophobic interior. Fluorescence quenching techniques have been employed to study apoLp-III helix topography and spatial arrangement in phospholipid disc complexes and intact lipoprotein particles. Intrinsic fluorescence of the single tyrosine in apoLp-III was exploited to monitor the location of helix 5 in model disc complexes. To investigate other regions of the protein, site-directed mutagenesis was performed to introduce cysteine residues, replacing Asn-40 (helix 2, N40C) or Leu-90 (helix 3, L90C), thereby providing two mutant apoLp-IIIs, each with a single site for covalent attachment of the extrinsic fluorescent probe, N-(1-pyrene) maleimide. In the lipid-free state, pyrene-N40C- and pyrene-L90C-apoLp-III were highly accessible to the negatively charged aqueous quencher KI, yieldingK sv values of 27.1 and 19.8m −1, respectively. Upon binding to the surface of a spherical lipoprotein particle, K sv values for KI decreased by about 90% for both pyrene-labeled apoLp-IIIs, indicating a significant change in the local microenvironment of the fluorophores. A lesser decrease in K sv was observed when the pyrene-labeled apoLp-IIIs were bound to phospholipid disc complexes. When spin-labeled fatty acids 5-doxylstearic acid and 12-doxylstearic acid were used as lipophilic quenchers, tyrosine and pyrene fluorescence were more effectively quenched by 5-doxylstearic acid in both phospholipid bilayer disc complexes and spherical lipoprotein particles. These data provide insight into the spatial topography of apoLp-III α-helices in phospholipid disc complexes and support the concept that interaction with spherical lipoprotein particles results in superficial contact of apoLp-III helical segments with the monolayer surface, providing a basis for its reversible binding ability.

Metabolic relevance of selenium in the insectCorcyra cephalonica

Requirement, uptake, and subcellular distribution of Na275SeO3 in the larvae of the insectC. cephalonica was investigated. That Se is well tolerated byC. cephalonica upto an added level of 2 ppm in the diet is suggested by the observed increase in body weight, total protein, and succinate dehydrogenase levels. Significant increases in the State 3 respiration ensued with Se supplementation up to 2 ppm in the mitochondrial oxidation of D-glycerol 1-phosphate, succinate and NADH, along with concomitant unaltered State 4 respiration, leading to enhanced RCR values. Maximal uptake of75Se was registered in the larvae maintained on basal diet when subjected to short-term exposure to 0.5 ppm75Se level. When exposure level was further increased up to 20 ppm, the observed decrease in the uptake of75Se suggested that Se status of larvae itself controlled the tissue uptake. Subcellular distribution pattern revealed maximal incorporation of75Se (cpm/g tissue) in the supernatant fraction, whereas, maximal specific75Se activity (cpm/mg protein) was associated with the mitochondrial fraction. Autoradiography of the soluble fractions indicated the presence of single selenoprotein in the larval group with short term 2 ppm75Se exposure. Inherent Se controls both the extent and the nature of distribution of mitochondrial75Se incorporation. Uptake of45Ca by the insect mitochondria was enhanced by dietary Se up to 2 ppm but was unaffected by addition ofin vitro75Se in the medium. A more fundamental role for Se in the mitochondrial energy metabolism emerges from these studies.