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• W2016579573 abstract "The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration (≈90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75m (≈80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease. The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration (≈90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75m (≈80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease. apolipoprotein Fourier transform infrared spectroscopy dynamic light scattering dimyristoylphosphatidylcholine hydrodynamic radius molecular mass Apolipoprotein (apo)1 E plays a key role in lipid transport throughout the body including the nervous system and is involved in the maintenance and repair of neurons (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3353) Google Scholar, 2Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (275) Google Scholar). One of the common human apoE isoforms, apoE4, is a major risk factor for Alzheimer's disease (3Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1977-1981Crossref PubMed Scopus (3657) Google Scholar, 4Corder E.H. Saunders A.M. Strittmatter W.J. Schmechel D.E. Gaskell P.C. Small G.W. Roses A.D. Haines J.L. Pericak-Vance M.A. Science. 1993; 261: 921-923Crossref PubMed Scopus (7163) Google Scholar, 5Saunders A.M. Strittmatter W.J. Schmechel D., St. George-Hyslop P.H. Pericak-Vance M.A. Joo S.H. Rosi B.L. Gusella J.F. Crapper-MacLachlan D.R. Alberts M.J. Hulette C. Crain B. Goldgaber D. Roses A.D. Neurology. 1993; 43: 1467-1472Crossref PubMed Google Scholar) and atherosclerosis (6Utermann G. Hardewig A. Zimmer F. Hum. Genet. 1984; 65: 237-241Crossref PubMed Scopus (108) Google Scholar, 7Luc G. Bard J.-M. Arveiler D. Evans A. Cambou J.-P. Bingham A. Amouyel P. Schaffer P. Ruidavets J.-B. Cambien F. Fruchart J.-C. Ducimetiere P. Arterioscler. Thromb. 1994; 14: 1412-1419Crossref PubMed Google Scholar, 8Eichner J.E. Kuller L.H. Orchard T.J. Grandits G.A. McCallum L.M. Ferrell R.E. Neaton J.D. Am. J. Cardiol. 1993; 71: 160-165Abstract Full Text PDF PubMed Scopus (232) Google Scholar). ApoE4 is also associated with poor recovery from head injury and stroke (9Mayeux R. Ottman R. Maestre G. Ngai C. Tang M.-X. Ginsberg H. Chun M. Tycko B. Shelanski M. Neurology. 1995; 45: 555-557Crossref PubMed Scopus (513) Google Scholar, 10Slooter A.J.C. Tang M.-X. van Duijn C.M. Stern Y. Ott A. Bell K. Breteler M.M.B. Van Broeckhoven C. Tatemichi T.K. Tycko B. Hofman A. Mayeux R. J. Am. Med. Assoc. 1997; 277: 818-821Crossref PubMed Google Scholar, 11Teasdale G.M. Nicoll J.A.R. Murray G. Fiddes M. Lancet. 1997; 350: 1069-1071Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), cognitive decline associated with coronary bypass surgery (12Tardiff B.E. Newman M.F. Saunders A.M. Strittmatter W.J. Blumenthal J.A. White W.D. Croughwell N.D. Davis R.D., Jr. Roses A.D. Reves J.G. the Neurologic Outcome Research Group of the Duke Heart Center Ann. Thorac. Surg. 1997; 64: 715-720Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), increased severity of tissue damage in multiple sclerosis (13Fazekas F. Strasser-Fuchs S. Schmidt H. Enzinger C. Ropele S. Lechner A. Flooh E. Schmidt R. Hartung H.-P. J. Neurol. Neurosurg. Psychiatry. 2000; 69: 25-28Crossref PubMed Scopus (82) Google Scholar), shortening of survival after the onset of amyotrophic lateral sclerosis (14Drory V.E. Birnbaum M. Korczyn A.D. Chapman J. J. Neurol. Sci. 2001; 190: 17-20Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and a poor response to other forms of central nervous system stress (15Roses A.D. Ann. N. Y. Acad. Sci. 1998; 855: 738-743Crossref PubMed Scopus (73) Google Scholar).The three common isoforms of apoE (apoE2, apoE3, and apoE4) are genetically determined and differ in cysteine and arginine content at positions 112 and 158: apoE2 (Cys112, Cys158), apoE3 (Cys112, Arg158), and apoE4 (Arg112, Arg158) (16Weisgraber K.H. Rall S.C., Jr. Mahley R.W. J. Biol. Chem. 1981; 256: 9077-9083Abstract Full Text PDF PubMed Google Scholar, 17Rall S.C., Jr. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1982; 257: 4171-4178Abstract Full Text PDF PubMed Google Scholar). The protein contains two distinct structural domains: a 22-kDa amino-terminal domain and a 10-kDa carboxyl-terminal domain (18Aggerbeck L.P. Wetterau J.R. Weisgraber K.H., Wu, C.-S.C. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar, 19Wetterau J.R. Aggerbeck L.P. Rall S.C., Jr. Weisgraber K.H. J. Biol. Chem. 1988; 263: 6240-6248Abstract Full Text PDF PubMed Google Scholar). In apoE4 and not the other isoforms, the two domains interact in a unique manner. In apoE4, Arg112 causes Arg61 to assume a unique conformation and interact with Glu255 in the carboxyl-terminal domain. This novel property of apoE4 is referred to as apoE4 domain interaction (20Dong L.-M. Wilson C. Wardell M.R. Simmons T. Mahley R.W. Weisgraber K.H. Agard D.A. J. Biol. Chem. 1994; 269: 22358-22365Abstract Full Text PDF PubMed Google Scholar, 21Dong L.-M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and was suggested to contribute to the association of apoE4 with disease (2Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (275) Google Scholar,21Dong L.-M. Weisgraber K.H. J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar).Previously, we demonstrated that the two domains of apoE unfold independently for all three isoforms (19Wetterau J.R. Aggerbeck L.P. Rall S.C., Jr. Weisgraber K.H. J. Biol. Chem. 1988; 263: 6240-6248Abstract Full Text PDF PubMed Google Scholar, 22Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (262) Google Scholar) and that the 22-kDa fragments, which contain the amino acid interchanges, differ in their susceptibility to thermal and chemical denaturation (apoE4 < apoE3 < apoE2) (22Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (262) Google Scholar, 23Acharya P. Segall M.L. Zaiou M. Morrow J. Weisgraber K.H. Phillips M.C. Lund-Katz S. Snow J. Biochim. Biophys. Acta. 2002; 1584: 9-19Crossref PubMed Scopus (63) Google Scholar). Denaturation of apoE2 with guanidine at neutral pH displayed two-stage cooperative unfolding, whereas apoE3 and apoE4 displayed noncooperative unfolding that was much more prominent with apoE4. This noncooperative unfolding of apoE4 suggested the presence of a stable folding intermediate (22Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (262) Google Scholar).Folding intermediates that are both stable under certain conditions and have nearly native structural features are referred to as molten globules (24Ptitsyn O.B. Adv. Protein Chem. 1995; 47: 83-229Crossref PubMed Google Scholar). Three structural features characterize the molten globule state. First, a significant amount of secondary structure of the native state is retained. Second, although there is considerable loss of tertiary structure, the molten globule is structurally compact. Third, there is internal mobility with exposure of the hydrophobic core. Until recently, it was assumed that the molten globule was a relatively rare state for a protein. However, there is increasing evidence that molten globules are common and that they play a key role in a wide variety of physiological processes, including translocation across membranes, increased affinity for membranes, binding to liposomes and phospholipids, protein trafficking, extracellular secretion, and the control and regulation of the cell cycle (24Ptitsyn O.B. Adv. Protein Chem. 1995; 47: 83-229Crossref PubMed Google Scholar, 25Dobson C.M. Philos. Trans. R. Soc. Lond-Biol. Sci. 2001; 356: 133-145Crossref PubMed Scopus (767) Google Scholar). Indeed, apolipoproteins, including human apoA-I and insect apolipophorin III, have also been reported as molten globules (26Gursky O. Atkinson D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2991-2995Crossref PubMed Scopus (172) Google Scholar, 27Weers P.M.M. Kay C.M. Ryan R.O. Biochemistry. 2001; 40: 7754-7760Crossref PubMed Scopus (26) Google Scholar). In these cases, it was proposed that internal mobility provides structural plasticity for binding to lipoprotein surfaces (26Gursky O. Atkinson D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2991-2995Crossref PubMed Scopus (172) Google Scholar, 27Weers P.M.M. Kay C.M. Ryan R.O. Biochemistry. 2001; 40: 7754-7760Crossref PubMed Scopus (26) Google Scholar).In this report, we demonstrate that apoE4 forms a stable folding intermediate more readily than apoE3 and apoE2. Using a variety of structural tools to characterize its structure, including pepsin proteolysis, Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS), we show that this stable apoE4 folding intermediate possesses the structural characteristics of a molten globule. We conclude that, in addition to apoE4 domain interaction, the propensity of apoE4 to form a molten globule may contribute to its association with disease.RESULTSTo follow up on our previous guanidine denaturation studies that suggested the presence of an apoE4 folding intermediate (22Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (262) Google Scholar), the 22-kDa fragments of apoE3 and apoE4 were examined by urea denaturation at pH 7.4 and pH 4.0 since low pH facilitates the formation of stable folding intermediates (molten globules). The denaturation curves at pH 7.4 reflected an apparent two-state denaturation. The midpoints of denaturation for the 22-kDa fragments of apoE3 and apoE4 were 5.2 and 4.3 m urea, respectively, consistent with previous results (22Morrow J.A. Segall M.L. Lund-Katz S. Phillips M.C. Knapp M. Rupp B. Weisgraber K.H. Biochemistry. 2000; 39: 11657-11666Crossref PubMed Scopus (262) Google Scholar) (Fig. 1 A). At pH 4.0, apoE4 and apoE3 displayed the same order of denaturation (apoE4 > apoE3). However, there was a distinct plateau in the curves for both isoforms, suggesting the presence of a stable folding intermediate (Fig. 1 B). As with guanidine denaturation, apoE2 was the most resistant to unfolding in urea and lacked an obvious plateau indicating that it did not form a folding intermediate (Fig. 1 B.) The data in Fig. 1 B were fitted to a 2-state model (unfolded/folded, solid lines overlaying the data). The poor fits to the apoE3 and apoE4 data further highlight the presence of stable folding intermediates in comparison to the reasonable fit obtained for the apoE2 data. Therefore, the data were analyzed according to a three-state model (native/intermediate/unfolded) (28Barrick D. Baldwin R.L. Biochemistry. 1993; 32: 3790-3796Crossref PubMed Scopus (232) Google Scholar), which gave excellent fits for the apoE3 and apoE4 isoforms (Fig. 1 C) but did not give a better fit for apoE2 than the two-state model. Fig. 2 shows the fractions of folded, intermediate, and unfolded protein for apoE3 and apoE4, according to the three-state model. The concentration of urea at which the folding intermediate was at maximum concentration was 3.75 m for the apoE4 22-kDa fragment (≈90%) and 4.75 m for the apoE3 fragment (≈80%). These results demonstrate that in urea the folding intermediate is a stable thermodynamic state, the first criterion for a molten globule.Figure 2Urea denaturation curves of the 22-kDa fragments of apoE3 and apoE4 at pH 4. The curves were analyzed by using a three-state model to determine the fraction of native (solid line), intermediate (dashed line), and unfolded (dotted line) structures at various urea concentrations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Pepsin ProteolysisSince proteolysis is a sensitive probe for conformational changes in proteins (33Spolaore B. Bermejo R. Zambonin M. Fontana A. Biochemistry. 2001; 40: 9460-9468Crossref PubMed Scopus (42) Google Scholar), the apoE fragments were subjected to limited proteolysis with pepsin at low pH with or without urea and analyzed by SDS-PAGE and amino-terminal sequencing. In 0m urea, there was one major fragment, which had the amino-terminal sequence of RQQTE, which corresponds to amino acids 15–19 in apoE (Fig. 3, top panels). This sequence is at the flexible amino terminus of the 22-kDa fragment that is not resolved in the x-ray structure (34Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (595) Google Scholar). Further addition of pepsin or longer digestion times did not produce smaller cleavage products under these conditions.Figure 3Pepsin proteolysis as a probe of the conformation of apoE3 and apoE4. The 22-kDa fragments of apoE3 and apoE4 were subjected to limited pepsin digestion in the presence and absence of urea at pH 4.0, and the extent of fragmentation was monitored by SDS-PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Digestion of the apoE4 22-kDa fragment in 3.75 m urea, the concentration at which the intermediate represents ≈90% of the mixture, revealed seven major bands (Fig. 3, middle panels). Bands 1–5 had the amino-terminal sequence GS1KVE, the same as that of recombinant apoE (it contains the novel Gly-Ser sequence at the amino terminus) (35Morrow J.A. Arnold K.S. Weisgraber K.H. Protein Expr. Purif. 1999; 16: 224-230Crossref PubMed Scopus (91) Google Scholar). Band 4 also contained a fragment with the amino-terminal sequence 79EEQLTP. Band 6 had the amino-terminal sequence 122VQYRG. Band 7 was rather broad and contained three fragments (124AMLGQSTEE,133RVRLASHLR, and 116VQYRGEVQA). Digestion of the apoE3 22-kDa fragment in 3.75 m urea yielded the same bands as the apoE4 digestion but with less proteolysis of the intact apoE3 fragment (Fig. 3, middle panels).The bands after digestion of apoE3 and apoE4 in 4.75 m urea were similar to those obtained after digestion in 3.75 murea, but there was less difference in the extent of digestion (Fig. 3,bottom panels). This result is consistent with the prediction, based on analysis of a three-state model, that similar amounts of the intermediate states from each isoform would be present in 4.75 m urea but not in 3.75 m urea. The increased sensitivity to pepsin digestion is also consistent with an altered conformation at low pH in the presence of urea, another characteristic of a molten globule. It is also important to note that there are a limited number of exposed pepsin cleavage sites, which is consistent with a limited structural or conformational reorganization of the apoE4 intermediate without complete loss of native structure.FTIRSince it is difficult to accurately estimate the secondary structure of a protein in urea by far ultraviolet circular dichroism due to the high absorbance of urea below 210 nm, we used a novel FTIR method to assess the secondary structure of the intermediate in urea. This method includes the subtraction of the urea background, as well as subtraction of absorbed (partially denatured) protein (29Oberg K.A. Fink A.L. Anal. Biochem. 1998; 256: 92-106Crossref PubMed Scopus (135) Google Scholar). The apoE4 22-kDa fragment was analyzed at pH 4.0 in the presence or absence of 3.75 m urea (Fig. 4). ApoE4 22-kDa in 0 m urea displayed 75% α-helix and 3% β-sheet, consistent with the α-helical content estimated by circular dichroism (18Aggerbeck L.P. Wetterau J.R. Weisgraber K.H., Wu, C.-S.C. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar) and x-ray crystallography (34Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (595) Google Scholar). In 3.75 m urea, apoE4 22-kDa displayed 46% α-helix and 17% β-sheet. Thus, the intermediate retains 61% of the native helical content, another criterion of a molten globule. In addition, it has a significant increase in β structure, which has implications for promoting aggregation and fibrillization.Figure 4FTIR analysis of apoE4 22-kDa fragment.Analysis in the amide I and II regions of the spectra was performed in the absence and presence of urea. The analysis in the absence of urea represents a single determination; the estimate of 75% α-helix is in excellent agreement with the x-ray crystal structure (34Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (595) Google Scholar). The analysis in the absence of urea was performed in duplicate with results in 3% agreement.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DLSDLS was used to determine the aggregation state of the intermediate. The measured hydrodynamic radii and estimated molecular masses are summarized in Table I. The shape-corrected M r calculated for the reference sample apoE4 22-kDa fragment at pH 7.4 with no urea was 22 kDa. At pH 4.0 (no urea), the size distribution (polydispersity) was wider, and the R h was larger. Although the difference was not significant within the error of the experiment, it is reasonable to speculate that both the larger R h and the greater size distribution indicate a somewhat lower stability of the apoE4 22-kDa fragment at the acidic pH, consistent with its increased tendency to form an intermediate at pH 4.0. A small widening in the flexible and dynamic helix bundle, as indicated by crystallographic studies (36Segelke B.W. Forstner M. Knapp M. Trakhanov S.D. Parkin S. Newhouse Y.M. Bellamy H.D. Weisgraber K.H. Rupp B. Protein Sci. 2000; 9: 886-897Crossref PubMed Scopus (47) Google Scholar), would not lead to a change in the helical content as determined from circular dichroism spectra and thus would still be compatible with a small increase in the R h, indicating a flexing of the four-helix bundle at pH 4.0.Table IHydrodynamic radius and derived properties for the apoE4 22-kDa fragmentpH 7.4 4.0 4.0[urea] (M) 0.0 0.0 3.75Hydrodynamic radius (nm, e.s.u.)2.40 ± 0.472.61 ± 0.673.93 ± 0.4Polydispersity (%)1-aPolydispersity is a measure of the size distribution.19.525.512.5M r (kDa, globular estimate)26.131.782.8M r(kD,1-aPolydispersity is a measure of the size distribution. shape corrected)1-bProlate ellipsoid shape correction factor f = 1.08 used in calculation for columns 1 and 2. A random coil model was used for the calculation of M r in column 3.21.826.524.21-a Polydispersity is a measure of the size distribution.1-b Prolate ellipsoid shape correction factor f = 1.08 used in calculation for columns 1 and 2. A random coil model was used for the calculation of M r in column 3. Open table in a new tab A more dramatic change was observed in the hydrodynamic behavior of the apoE4 22-kDa fragment at pH 4.0 in 3.75 m urea.R h increased significantly, but the size distribution remained narrow, indicating a well-defined intermediate species. Assuming a large contribution of random coil conformation in the intermediate, the M r corresponding to theR h for a random coil model was estimated to be ≈24 kDa, consistent with a monomeric species and no evidence of aggregation under these conditions.Lipid Binding Abilities of the Three IsoformsSince molten globules have been implicated in membrane association and phospholipid binding (37Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), the relative abilities of the three isoforms to bind and disrupt DMPC vesicles were determined at pH 4.0 in a turbidimetric clearing assay under urea concentrations where the intermediate species is highly populated for apoE4 and apoE3. It is important to note that while the carboxyl-terminal domain of apoE contains the major lipid binding determinants, the N-terminal 22-kDa domain also is capable of binding to lipid. Previous studies have indicated that the N-terminal 22-kDa fragment clears at approximately half the rate of the intact protein at pH 7.4 (38Segall M.L. Dhanasekaran P. Baldwin F. Anantharamaiah G.M. Weisgraber K.W. Phillips M.C. Lund-Katz S. J. Lipid Res. 2002; 43: 1688-1700Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In the presence of 3.75m urea, where the apoE4 22-kDa fragment has its maximum population of intermediate species (≈90%), apoE4 is more effective in clearing DMPC solutions than both apoE3 and apoE2 (Fig. 5). In 4.75 m urea, where apoE3 has its maximum population of intermediate species (≈80%) and apoE4 is close to its maximum population (≈80%), apoE3 and apoE4 have a similar rate of clearance, while apoE2 lags behind. At 4.75m urea, the relative clearance rate of apoE2 is closer to that of apoE4 and apoE3 than at 3.75 m urea. There are two reasons for this. First, the DMPC vesicles are smaller at 4.75m urea than at 3.75 m, based on their relative scattering intensities. Thus, the lipid substrate is different at the two urea concentrations. Second, at 4.75 m urea, the apoE2 is beginning to unfold, which would be predicted to increase its lipid-binding ability. The important point is that apoE2 still lags behind apoE4 and apoE3, which is consistent with its greater stability and absence of any significant concentration of a folding intermediate. Overall, the results are consistent with the enhanced ability of the intermediate species to remodel DMPC compared with the folded state.Figure 5Comparison of the lipid-binding activities of the apoE2, apoE3, and apoE4 22-kDa fragments at pH 4.0. The relative abilities of the fragments to clear DMPC vesicles were determined in a turbidimetric clearing assay in the presence of 3.75 and 4.75 m urea where the population of intermediate species is highest for apoE3 and apoE4, respectively. Clearance curves are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThis study shows that the folding intermediate of apoE4 can be stabilized and its structure characterized at pH 4.0 in 3.75m urea. Pepsin digestion revealed that, in forming the intermediate, apoE4 undergoes a conformational change that involves opening of the four-helix bundle. FTIR analysis demonstrates that the intermediate retains much of its secondary structure (61%), with a modest increase in β structure. The DLS results indicate that the intermediate is a single molecule with a narrow polydispersity and slightly elongated structure. These structural properties of the apoE4 intermediate are consistent with those of a molten globule. Based on our structural characterization, we propose a model for the apoE4 molten globule (Fig. 6). The bundle is partially open, generating a slightly elongated structure and exposing the hydrophobic core (DLS and pepsin digestion data), and most of the α-helical structure is retained (FTIR and pepsin cleavage data). We suggest that the helical structure is lost at the end of the helices where it is converted to β structure or random structure, exposing the pepsin cleavage sitesFigure 6Model of the apoE4 22-kDa fragment in its molten globule state. Based on the structural and physical characterization of the apoE4 molten globule, we propose that at pH 4.0 the four-helix bundle of apoE is partially open, generating a slightly elongated structure in which there is a shortening of the α-helices and an increase in β structure at the ends of the helices. The peptic cleavage sites that are exposed in the molten globule state (Fig. 3) are indicated by the small arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The largely opened conformation of the four-helix bundle in the intermediate is similar to the conformation change of apoE when it binds to lipid (36Segelke B.W. Forstner M. Knapp M. Trakhanov S.D. Parkin S. Newhouse Y.M. Bellamy H.D. Weisgraber K.H. Rupp B. Protein Sci. 2000; 9: 886-897Crossref PubMed Scopus (47) Google Scholar, 37Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 39Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 40Fisher C.A. Narayanaswami V. Ryan R.O. J. Biol. Chem. 2000; 275: 33601-33606Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The DLS data are consistent with an intermediate consisting of short (≈20 Å) helical segments or building blocks of the stable domain, tethered by long segments containing random coil and β structure. Although it is difficult to estimate accurately the hydrodynamic parameters of the intermediate model, it is reasonable to assume that such a chain of tethered helical segments would display very much the behavior we observe for the folding intermediate.The emerging view is that molten globules play an important role in many physiological processes, including translocation across membranes, increased affinity for membranes, binding to liposomes and phospholipids, protein trafficking, extracellular secretion, and the control and regulation of the cell cycle (24Ptitsyn O.B. Adv. Protein Chem. 1995; 47: 83-229Crossref PubMed Google Scholar, 25Dobson C.M. Philos. Trans" @default.
• W2016579573 created "2016-06-24" @default.
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• W2016579573 date "2002-12-01" @default.
• W2016579573 modified "2023-10-17" @default.
• W2016579573 title "Apolipoprotein E4 Forms a Molten Globule" @default.
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