Today the determination of successful crystallization conditions for a particular macromolecule remains a highly empirical process. Sparse-matrix and grid-screening procedures are rapid and economical means to determine preliminary crystallization conditions. During optimization the variable set (pH, precipitant type and precipitant concentration) utilized in these procedures is screened in an attempt to determine appropriate conditions for the nucleation and growth of single crystals suitable for X-ray diffraction analysis. Unfortunately, in many cases this strategy will not produce single crystals suitable for X-ray diffraction analysis. We have explored, in an empirical sense, other tools for use during optimization. First, a new screening protocol is evaluated which employs less classical precipitating agents. Second, a set of 24 electrostatic crosslinking agents are evaluated for their ability to promote crystallization. Third, a panel of more than 30 detergents are evaluated for their ability to prevent sample aggregation and influence crystal growth.

SUMMARY Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function on proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies (IBs), IB formation may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that monoclonal antibody (mAb) 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular weight conformational states expanded polyQ assumes and disappears in higher molecular-weight aggregated forms, such as IBs. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE•phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4•palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4•POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.

We assessed the isoform-specific effects of apolipoprotein (apo) E on the response of Neuro-2a cells to the amyloid ␤ peptide (A␤1-42). As determined by the intracellular staining pattern and the release of ␤-hexosaminidase into the cytosol, apoE4-transfected cells treated with aggregated A␤1-42 showed a greater tendency toward lysosomal leakage than neo-or apoE3-transfected cells. A␤1-42 caused significantly greater cell death and more than 2-fold greater DNA fragmentation in apoE4-secreting than in apoE3-secreting or control cells. H 2 O 2 or staurosporine enhanced cell death and apoptosis in apoE4-transfected cells but not in apoE3-transfected cells. A caspase-9 inhibitor abolished the potentiation of A␤1-42-induced apoptosis by apoE4. Similar results were obtained with conditioned medium from cells secreting apoE3 or apoE4. Cells preincubated for 4 h with a source of apoE3 or apoE4, followed by removal of apoE from the medium and from the cell surface, still exhibited the isoform-specific response to A␤1-42, indicating that the potentiation of apoptosis required intracellular apoE, presumably in the endosomes or lysosomes. Studies of phospholipid (dimyristoylphosphatidylcholine) bilayer vesicles encapsulating 5-(and-6)-carboxyfluorescein dye showed that apoE4 remodeled and disrupted the phospholipid vesicles to a greater extent than apoE3 or apoE2. In response to A␤1-42, vesicles containing apoE4 were disrupted to a greater extent than those containing apoE3. These findings are consistent with apoE4 forming a reactive molecular intermediate that avidly binds phospholipid and may insert into the lysosomal membrane, destabilizing it and causing lysosomal leakage and apoptosis in response to A␤1-42.Human apolipoprotein (apo) 1 E exists in three major isoforms, apoE2, apoE3, and apoE4, which are encoded by three apoE alleles (⑀2, ⑀3, and ⑀4) (1). The ⑀4 allele is a susceptibility gene for Alzheimer's disease (2-5) and other neurodegenerative disorders (6 -9). The apoE isoforms have differential effects on neurite outgrowth in cultured neurons and on the stability of the intracellular cytoskeleton and microtubular system (10 -14). Recently, we demonstrated that a bioactive form of apoE (E4 to a greater extent than E3) can escape an intracellular membrane compartment, enter the cytosol, and interact with cytoskeletal components to form neurofibrillary tangle-like neuronal inclusions (15). Our studies in transgenic mice have indicated a role for apoE4 in neuronal degeneration and behavioral activity (16,17). In addition, apoE is critical in the deposition of amyloid ␤ peptide (A␤) in transgenic mice overproducing the amyloid precursor protein (18 -20).The neurotoxicity of A␤1-42 appears to be an important factor in the pathogenesis of Alzheimer's disease (for review, see Refs. 21 and 22). A␤1-42 is a proteolytic product of the amyloid precursor protein and is a normal component of plasma and cerebrospinal fluid (23,24). A␤ deposition or amyloid plaque formation is found in Alzheimer's disease cases (25,26) and in t...

Apolipoprotein (apo)E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 Å resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex.

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural study of full-length apoE is hindered by apoE's oligomerization property. Using protein-engineering techniques, we generated a monomeric, biologically active, full-length apoE. Cross-linking experiments indicate that this mutant is nearly 95-100% monomeric even at 20 mg/mL. CD spectroscopy and guanidine hydrochloride denaturation demonstrate that the structure and stability of the monomeric mutant are identical to wild-type apoE. Monomeric and wild-type apoE display similar lipid-binding activities in dimyristoylphosphatidylcholine clearance assays and formation of reconstituted high-density lipoproteins. Furthermore, the monomeric and wild-type apoE proteins display an identical LDL receptor binding activity. Availability of this monomeric, biologically active, full-length apoE allows us to collect high quality NMR data for structural determination. Our initial NMR data of lipid-free apoE demonstrates that the N-terminal domain in the full-length apoE adopts a nearly identical structure as the isolated N-terminal domain, whereas the C-terminal domain appears to become more structured than the isolated C-terminal domain fragment, suggesting a weak domain-domain interaction. This interaction is confirmed by NMR examination of a segmental labeled apoE, in which the N-terminal domain is deuterated and the C-terminal domain is double-labeled. NMR titration experiments further suggest that the hinge region (residues 192-215) that connects apoE's N- and C-terminal domains may play an important role in mediating this domain-domain interaction.

The defective binding of apolipoprotein (apo) E2 to lipoprotein receptors, an underlying cause of type III hyperlipoproteinemia, results from replacement of Arg 158 with Cys, disrupting the naturally occurring salt bridge between Asp 154 and Arg 158. A new bond between Asp 154 and Arg 150 is formed, shifting Arg 150 out of the receptor binding region. Elimination of the 154-150 salt bridge by site-directed mutagenesis of Asp 154 to Ala restored the receptor binding activity to near normal levels. The X-ray crystal structure of apoE2 Ala 154 demonstrated that Arg 150 was relocated within the receptor binding region. Our results demonstrate that defective binding of apoE2 occurs by a novel mechanism of the replacement of one salt bridge with another.

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, 13C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N-and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the ␣-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N-and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N-and O-sulfo groups such as that found in the liver and the brain. Human apolipoprotein E (apoE)1 is a 299-residue polymorphic protein that facilitates the transport and metabolism of lipids (1). ApoE is a ligand for members of the low density lipoprotein (LDL) receptor family, heparin, and heparan sulfate proteoglycans (HSPGs) (2, 3). It is composed of two domains: a 22-kDa NH 2 -terminal domain (residues 1-191) and a 10-kDa COOH-terminal domain (residues 216 -299) (4). The 22-kDa NH 2 -terminal domain contains the primary HSPGbinding site (residues 140 -150) (5) colocalized with the LDL receptor binding site (6 -8).Binding of apoE to HSPG is an initial step in the localization of apoE-containing lipoproteins to the surface of several different types of cells (9). After localization, the apoE-containing lipoproteins are transported into the cell by pathways dependent on either the LDL receptor or the LDL receptor-related protein (LRP) or by direct uptake of an apoE-containing lipoprotein-HSPG complex (10). Binding of apoE to HSPG affects neurite extension in neurons (11, 12) and localizes secreted apoE to the surface of macrophages (13). Binding of apoE to HSPG may also play a role in Alzheimer's disease through either competition between apoE and the amyloid precursor protein (APP) for HSPG-binding sites or by modulation of the HSPG/LRP uptake pathway (14 -17).The best understood physiological role of the binding of apoE to HSPG is in lipoprotein remnant clearance....