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• W1976501903 abstract "Apolipoprotein E (apoE) is important in lipid metabolism due to its interaction with members of the low density lipoprotein (LDL) receptor family. ApoE is able to interact with the LDL receptor only when it is bound to lipid particles. To address structural aspects of this phenomenon, a receptor-active apoE peptide, encompassing the receptor-binding region of the protein, was studied by NMR in the presence of the lipid-mimicking agent trifluoroethanol. In 50% trifluoroethanol, apoE-(126–183) forms a continuous amphipathic α-helix over residues Thr130–Glu179. Detailed NMR relaxation analysis indicates a high degree of plasticity for the residues surrounding 149–159. This intrinsic flexibility imposes a curvature to the peptide that may be important in terms of interaction of apoE with various sized lipid particles and the LDL receptor. Residues 165–179 of apoE may act as a molecular switch whereby these residues are unstructured in the absence of lipids and prevent interaction with the LDL receptor. In the presence of lipids, these residues become helical resulting in a receptor-active conformation of the protein. Furthermore, the electrostatic characteristics and geometric features of apoE-(126–183) suggest that apoE binds to the LDL receptor by interacting with more than one of the receptor ligand-binding repeats. Apolipoprotein E (apoE) is important in lipid metabolism due to its interaction with members of the low density lipoprotein (LDL) receptor family. ApoE is able to interact with the LDL receptor only when it is bound to lipid particles. To address structural aspects of this phenomenon, a receptor-active apoE peptide, encompassing the receptor-binding region of the protein, was studied by NMR in the presence of the lipid-mimicking agent trifluoroethanol. In 50% trifluoroethanol, apoE-(126–183) forms a continuous amphipathic α-helix over residues Thr130–Glu179. Detailed NMR relaxation analysis indicates a high degree of plasticity for the residues surrounding 149–159. This intrinsic flexibility imposes a curvature to the peptide that may be important in terms of interaction of apoE with various sized lipid particles and the LDL receptor. Residues 165–179 of apoE may act as a molecular switch whereby these residues are unstructured in the absence of lipids and prevent interaction with the LDL receptor. In the presence of lipids, these residues become helical resulting in a receptor-active conformation of the protein. Furthermore, the electrostatic characteristics and geometric features of apoE-(126–183) suggest that apoE binds to the LDL receptor by interacting with more than one of the receptor ligand-binding repeats. apolipoprotein E chemical shift index dodecylphosphocholine heteronuclear single quantum correlation low density lipoprotein very LDL nuclear Overhauser effect nuclear Overhauser effect enhancement spectroscopy trifluoroethanol LDL receptor ligand-binding repeats radians Human apolipoprotein E (apoE)1 plays an important role in lipid metabolism by stabilizing lipoprotein particles and regulating plasma triglyceride clearance and cholesterol homeostasis (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3336) Google Scholar, 2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar) through its interaction with members of the lipoprotein receptor family, such as the low density lipoprotein (LDL) receptor, the LDL receptor-related protein (3Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5810-5814Crossref PubMed Scopus (452) Google Scholar), and the very low density lipoprotein receptor (4Takahashi S. Kawarabayasi Y. Nakai Y. Saskai J. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9252-9256Crossref PubMed Scopus (474) Google Scholar). ApoE is also found associated with lipoproteins in cerebrospinal fluid (5Pitas R.E. Boyles J.K. Lee S.H. Hui D. Weisgraber K.H. J. Biol. Chem. 1987; 262: 14352-14360Abstract Full Text PDF PubMed Google Scholar) and is involved in nerve regeneration (6Handelmann G.E. Boyles J.K. Weisgraber K.H. Mahley R.W. Pitas R.E. J. Lipid Res. 1992; 33: 1677-1688Abstract Full Text PDF PubMed Google Scholar). The E4 isoform has been demonstrated as a major genetic risk in the predisposition of Alzheimer's disease (7Strittmatter 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 (3633) Google Scholar, 8Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (275) Google Scholar). ApoE is composed of two structurally and functionally independent domains (9Aggerbeck 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, 10Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. J. Biol. Chem. 1988; 263: 6240-6248Abstract Full Text PDF PubMed Google Scholar). The 10-kDa C-terminal domain exhibits a high affinity for lipids and is thought to be the primary site for binding of apoE on spherical lipoprotein particles (11Westerlund J.A. Weisgraber K.H. J. Biol. Chem. 1993; 268: 15745-15750Abstract Full Text PDF PubMed Google Scholar). The 22-kDa N-terminal domain contains the receptor-binding region of the protein. This region has been localized between residues 136 and 150 and is essentially made of basic amino acids whose chemical modifications render apoE non-receptor-competent (12Mahley R.W. Innerarity T.L. Pitas R.E. Weisgraber K.H. Brown J.H. Gross E. J. Biol. Chem. 1977; 252: 7279-7287Abstract Full Text PDF PubMed Google Scholar, 13Weisgraber K.H. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1978; 253: 9053-9062Abstract Full Text PDF PubMed Google Scholar). In the absence of lipids, neither the N-terminal domain nor the full-length apoE protein are recognized by the LDL receptor (14Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). The N-terminal domain has a lower affinity for plasma lipoproteins than the C-terminal domain; however, the N-terminal domain is able to bind phospholipid vesicles and transform them into discoidal complexes that bind the LDL receptor with a similar efficiency as the entire protein (15Innerarity T.L. Friedlander B.J. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. J. Biol. Chem. 1983; 258: 12341-12347Abstract Full Text PDF PubMed Google Scholar). The two domains are linked by a protease-sensitive hinge region that has had no function associated to date. The structure of the N-terminal domain in lipid-free solution was solved at high resolution by x-ray crystallography. This domain is an elongated four-helix bundle made of amphipathic helices with hydrophobic faces oriented toward the interior of the bundle (16Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (593) Google Scholar). It has been postulated that the N-terminal four-helix bundle undergoes a conformational change upon lipid binding to expose the hydrophobic side-chains of the protein that interact with the lipid surface (2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 17De Pauw M. Vanloo B. Weisgraber K.H. Rosseneu M. Biochemistry. 1995; 34: 10953-10966Crossref PubMed Scopus (54) Google Scholar,18Raussens V. Fisher C.A. Goormaghtigh E. Ryan R.O. Ruysschaert J.-M. J. Biol. Chem. 1998; 273: 25825-25830Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). We have described previously (19Raussens V. Mah M.K.H. Kay C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) the purification and characterization of a peptide encompassing the portion of apoE that contains the receptor-binding region, apoE-(126–183). In this report, the structure and dynamics of the receptor-active apoE peptide, as determined by NMR spectroscopy in presence of TFE, are shown. Results indicate that apoE-(126–183) consists of an extended amphipathic helix that runs almost the entire length of the sequence. Structural calculations, in addition to relaxation data, indicate that the helix shows a high degree of plasticity. The dynamics study reported here marks the first such study of an extended helix in solution. The implications of the flexibility and helical structure of apoE-(126–183) are discussed in terms of whole apoE in the presence of a wide range of lipoprotein particles in the bloodstream. Furthermore, this study emphasizes the requirement of lipids bound to apoE for proper LDL receptor recognition. For preparation of apoE-(126–183), human apoE3-(1–183) was cloned into a pET vector, expressed in Escherichia coli BL21 cells, and purified as described previously (20Fisher C.A. Wang J. Francis G.A. Sykes B.D. Kay C.M. Ryan R.O. Biochem. Cell Biol. 1997; 75: 45-53Crossref PubMed Scopus (40) Google Scholar). Unlabeled and uniformly 15N labeled apoE3-(1–183) was prepared using standard media (21Muchmore D.C. McIntosh L.P. Russell C.B. Anderson D.E. Dahlquist F.W. Methods Enzymol. 1989; 177: 44-73Crossref PubMed Scopus (473) Google Scholar). ApoE-(126–183) was prepared by cleavage of the purified recombinant apoE3-(1–183) by CNBr as described previously (19Raussens V. Mah M.K.H. Kay C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). NMR experiments were performed on ∼2 mm unlabeled or 15N-labeled apoE-(126–183) in 500 μl of 50% TFE-d 3, 40% H2O, 10% D2O, pH 3.3, containing 0.01% (w/v) NaN3and 0.25 mm of 2,2-dimethyl-2-silapentane-5-sulfonate as an internal chemical shift reference. NMR experiments were carried out at 30 °C on Varian INOVA 500-MHz and Unity 600-MHz NMR spectrometers. Data were processed using NMRPIPE (22Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11279) Google Scholar) and analyzed using NMRVIEW (23Johnson B.A. Blevins R.A. J. Biomol. NMR. 1994; 4: 603-614Crossref PubMed Scopus (2647) Google Scholar). Complete 1H and 15N spectral assignments of apoE-(126–183) were obtained using gradient-enhanced three-dimensional15N-edited total correlation spectroscopy (τmix 51.4 ms) and NOESY (τmix 50 ms) experiments to identify spin systems and inter-residue connectivities as described by Wüthrich (24Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar). Confirmation of side-chain assignment was obtained through the use of three-dimensional HNHB and two-dimensional natural abundance 13C HSQC spectra. 15N T1, T2, and heteronuclear NOE relaxation data were recorded at 30 °C on both 500- and 600-MHz spectrometers using the enhanced sensitivity gradient pulse sequences developed by Farrow et al. (25Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Forman-Kay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (1992) Google Scholar). The T1relaxation decay was sampled at 10 different time points on each spectrometer: 11.1, 111, 277.5, 444, 555, 666, 777, 888, 999, and 1110 ms. The T2 relaxation decay was sampled at different 10 time points: 16.6, 33.2, 49.8, 66.4, 83.0, 99.7, 116.3, 132.9, 149.5, and 166.1 ms on the 500-MHz spectrometer and 16.3, 32.6, 48.9, 65.2, 81.4, 97.7, 114.0, 130.3, 146.6, and 162.9 ms on the 600-MHz spectrometer. The exponential decay curves for T1 and T2 peak intensities were fit using the in-house written program xcrvfit. 2R. Boyko, unpublished data. {1H}-15N NOE values were obtained from the ratio of the peak intensity from proton-saturated and -unsaturated spectra. Reduced spectral density mapping was carried out as described by Farrow et al. (26Farrow N.A. Zhang O. Szabo A. Torchia D.A. Kay L.E. J. Biomol. NMR. 1995; 6: 153-162Crossref PubMed Scopus (454) Google Scholar). An ensemble of 388 apoE-(126–183) structures was computed from 525 distance restraints (203 intraresidue, 202 sequential, and 120 medium range (defined as 2 >= i − j <= 4)), and 76 dihedral angle restraints (46 φ, 30 ψ) starting with an extended chain using a simulated annealing protocol (27Nilges M. Clore M. Gronenborn A.M. FEBS Lett. 1988; 229: 317-324Crossref PubMed Scopus (767) Google Scholar, 28Nilges M. Gronenborn A.M. Brünger A.T. Clore M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (512) Google Scholar) in X-PLOR version 3.851 (29Brünger A.T. A System for X-ray Crystallography and NMR, X-PLOR Version 3.1. Yale University Press, New Haven, CT1992Google Scholar). Interproton distance restraints were derived from three-dimensional15N-edited NOESY experiments recorded with a τmix of 50 and 100 ms, as well as a two-dimensional homonuclear NOESY experiment in D2O recorded with a τmix of 150 ms. Distances were calibrated according to Slupsky and Sykes (30Slupsky C.M. Sykes B.D. Biochemistry. 1995; 34: 15953-15964Crossref PubMed Scopus (185) Google Scholar). φ backbone dihedral angles were calculated based on measured 3 J HN-Hα coupling constants in an HNHA experiment (31Bax A. Vuister G.W. Grzesiek S. Delaglio F. Wang A.C. Tschudin R. Zhu G. Methods Enzymol. 1994; 239: 79-105Crossref PubMed Scopus (377) Google Scholar) and the Karplus equation (32Karplus M. J. Am. Chem. Soc. 1963; 85: 2870-2871Crossref Scopus (2263) Google Scholar). ψ dihedral angle restraints were obtained from the ratio of thed Nα(i,i)/dαN(i− 1,i) in the three-dimensional 15N-edited NOESY spectrum (33Gagné S. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). The X-PLOR energies for the 100 lowest energy structures are as follows: E total = 64.8 ± 1.7,E bonds = 14.6 ± 0.8,E angles = 46.8 ± 1.3,E vdw = 0.4 ± 0.6,E noe = 2.4 ± 0.7, and Ecdih = 0.5 ± 0.2 kcal·mol−1. No distance or dihedral angle violation was greater than 0.2 Å or 2°, respectively. Root mean square deviations from idealized values are as follows: bonds = 0.0039 ± 0.0001 Å, angles = 0.4207 ± 0.0059°, and improper angles = 0.0094 ± 0.0013°. Families of structures were extracted from the ensemble of structures by superimposing the backbone of residues 149–159 of apoE-(126–183) using the program NMRCLUST (34Kelly A.L. Gardner S.P. Sutcliffe M.J. Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (402) Google Scholar). ApoE-(126–183) is a 58-residue peptide that includes a lipid-binding region as well as the LDL receptor-binding moiety of apoE. ApoE-(126–183) is insoluble in aqueous solution above pH 4. Below pH 4, the peptide does not appear structured in water; however, in the presence of either lipids, such as dodecylphosphocholine (DPC), or lipid-mimicking agents, such as trifluoroethanol (TFE), it displays a high (70–80%) helical content (19Raussens V. Mah M.K.H. Kay C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Previously, it was determined that 50% TFE or 13 mmDPC is sufficient to induce full structuring of this peptide (19Raussens V. Mah M.K.H. Kay C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Furthermore, functional characterization has shown that, in the presence of lipids, this peptide is able to interact with the LDL receptor (19Raussens V. Mah M.K.H. Kay C.M. Sykes B.D. Ryan R.O. J. Biol. Chem. 2000; 275: 38329-38336Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Fig.1 exhibits a two-dimensional1H-15N HSQC NMR spectrum of apoE-(126–183) illustrating the quality of the data obtained for this peptide at an NMR frequency of 500 MHz. For the 58-residue peptide, most resonances are clearly resolved. Four resonances overlap (Lys157 with Arg158 and Ile177 with Arg180); one resonance is very weak and close to another peak (Leu148), and three residues are missing (the two N-terminal residues, Leu126, Gly127, and the residue preceding the C-terminal proline, Gly182). Despite having a well resolved spectrum, normally indicative of a protein with a well defined tertiary structure, no long range distance restraints (i − j >= 5) could be found in an 15N-edited NOESY spectrum with a mixing time of 150 ms. The secondary structure of apoE-(126–183) was determined using NMR spectroscopy based upon NOE connectivities, the Hα NMR chemical shift index (CSI) (35Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1776) Google Scholar, 36Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (925) Google Scholar), and the ratio of the dNα/dαN NOEs (33Gagné S. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). A summary of these data are illustrated in Fig. 2. In general, helical secondary structure was defined as follows: 1) the presence of a dαN(i,i+3) NOE; 2) a dNα/dαN ratio >1; 3) a3 J HNHα <6 Hz; and 4) upfield shifted Hα NMR chemical shifts relative to random coil chemical shifts (negative CSI). When more than half of the available criteria were met for a particular residue and the flanking residues, the secondary structure was assigned as helical. Fig. 2 shows that apoE-(126–183) appears to be composed of a long α-helix spanning the sequence from Thr130 to Glu179, with the first and last four residues appearing unstructured. For residues Ala176 to Glu179, the dαN(i,i+3) NOEs could not be assigned due to ambiguities arising from an almost complete overlap of Ile177 and Arg180. Nevertheless, the3 J HNHα, dNα/dαN, and CSI values for these residues favor a helical conformation. Fig. 2 also shows evidence of the simultaneous presence of dαN(i,i+2)and dαN(i,i+4) NOEs for some residues along the helix. These data were collected from a short mixing time NOESY (50 ms) and thus are not likely due to spin diffusion. According to Wüthrich (24Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar), dαN(i,i+4) is characteristic of an α-helix, whereas dαN(i,i+2) is characteristic of a 310 helix. The simultaneous presence of both NOEs for the same residue may reflect some sort of internal flexibility. The three-dimensional structure of apoE-(126–183) was calculated from the NMR data as described under “Experimental Procedures.” An ensemble of 388 structures was computed, none of which contained distance restraint violations greater than 0.2 Å nor dihedral angle violations greater than 2°. The 100 structures with the lowest calculated total energy were subsequently selected for further consideration. According to PROCHECK-NMR (37Laskowski R.A. Rullman J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4283) Google Scholar), 99.90% of the non-glycine residues have (φ, ψ) angles in the most favored or the additionally allowed regions of the Ramachandran plot for these 100 structures (data not shown). Attempts to superimpose the final 100 structures revealed a rather poor convergence. In general, it is well known that proteins, nucleic acids, and polysaccharides are flexible in solution and can adopt multiple conformations. Such conformational flexibility has been shown to be important for biological function. Most structures solved by NMR are usually reported as ensembles of structures fulfilling the experimental restraints to a similar and high extent (38Abseher R. Horstink L. Hilbers C.W. Nilges M. Proteins Struct. Funct. Genet. 1998; 31: 370-382Crossref PubMed Scopus (60) Google Scholar). However, when the dynamics or motion in a protein is more extensive, conformational families are believed to be a more realistic representation of the protein structure (34Kelly A.L. Gardner S.P. Sutcliffe M.J. Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (402) Google Scholar, 38Abseher R. Horstink L. Hilbers C.W. Nilges M. Proteins Struct. Funct. Genet. 1998; 31: 370-382Crossref PubMed Scopus (60) Google Scholar, 39Brüschweiler R. Blackledge M. Ernst R.R. J. Biomol. NMR. 1991; 1: 3-11Crossref PubMed Scopus (152) Google Scholar, 40Görler A. Ulyanov N.B. James T.L. J. Biomol. NMR. 2000; 16: 147-164Crossref PubMed Scopus (23) Google Scholar). In the case of apoE-(126–183), it is apparent that there are several conformationally related subfamilies that all fulfill the experimental restraints after the simulated annealing calculations. To visualize the structural diversity of apoE-(126–183), conformationally related subfamilies of structures were extracted using the program NMRCLUST (34Kelly A.L. Gardner S.P. Sutcliffe M.J. Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (402) Google Scholar). For clustering, the backbone heavy atoms of residues 149–159, the central core region of the peptide displaying similar R2/R1ratios (see “Discussion” below), were superimposed, and clustering was done on the backbone heavy atoms of residues 132–170. In total, 13 subfamilies of structures were found. Among these, the first five contain 68 of the 100 structures. Each of the other classes contained 5 or fewer structures. The subfamilies could be further divided in two categories as follows: those with their hydrophobic residues on the convex face of the helix, and those with their hydrophobic residues on the concave face of the helix. The first three subfamilies (Fig.3 A) have their hydrophobic residues located on the concave portion of the helix and represent 45 of the 68 structures found in the first 5 subfamilies. The structures in each of the first 3 subfamilies are moderately well defined over the entire helical length with root mean square distributions about the mean coordinate positions of 1.2 Å for subfamily 1, 1.3 Å for subfamily 2, and 1.6 Å for subfamily 3 for backbone atoms of residues 134–168. However, these subfamilies have quite differing degrees of helix curvature. With the hydrophobic face of the helix pointing toward the reader, a van der Waals surface of the representative structure of subfamily 1 is shown in Fig. 3 B. A continuous and almost perfectly aligned surface of hydrophobic residues appears to cover the inside face of the apoE-(126–183) peptide in this conformation. Such a disposition of the hydrophobic residues is well suited for an interaction of this peptide with another hydrophobic surface such as a lipid particle. Arg134, close to the hydrophobic-hydrophilic interface, is the only non-hydrophobic residue in this face of the peptide. To gain insight into the motions of apoE-(126–183) in solution, longitudinal (T1) and transverse (T2) 15N NMR relaxation times as well as {1H}-15N heteronuclear NOEs were measured. The R1 (1/T1) and R2 (1/T2) relaxation rates as well as the heteronuclear NOEs at field strengths of 500 and 600 MHz are shown in Fig. 4. R1, R2, and the heteronuclear NOE are useful NMR parameters for probing backbone dynamics on several time scales. Picosecond to nanosecond time scale motions are reflected in all three parameters. Slower millisecond to microsecond time scale motions are manifested in the parameter R2. Of the 58 residues in apoE-(126–183), 50 were used in the backbone dynamics analysis excluding the four overlapping resonances, the three missing resonances (the two N-terminal residues, and the penultimate C-terminal residue) and the C-terminal proline. The 1st panel in Fig. 4 illustrates the R1relaxation rate of apoE-(126–183). For a rigid globular (and hence isotopically tumbling) protein with flexible ends, it is typically observed that the R1 relaxation rates decrease sharply from the flexible N terminus to a plateau value that is maintained for the majority of the protein, ending with a sharp increase for the flexible C-terminal residues. For apoE-(126–183), there appears to be a gradual decrease in the R1 relaxation rate up to approximately residue 149 followed by a gradual increase after approximately residue 159. The 2nd panel illustrates the R2 relaxation rates. For a globular protein, it is typically observed that the R2 relaxation rate will follow the opposite behavior of R1; R2 sharply rises followed by a plateau region and subsequently sharply falls due to flexibility at the N and C termini. For apoE-(126–183), the first and last four residues follow this pattern of the sharp rise and fall. However, for residues 130–148, R2 appears to rise gradually followed by a plateau region from 149–159 followed by a more rapid decrease for residues 160–178. The 3rd panel in Fig. 4 shows the {1H}-15N heteronuclear NOE for apoE-(126–183). For a rigid globular protein, the heteronuclear NOE should follow a similar pattern to R2. For apoE-(126–183), the NOE rises sharply for the first few residues, followed by a gradual increase up to residue 136. The NOEs stay constant up to residue 156 followed by a slight decreasing trend to residue 179. Often there is a region such as a loop that exhibits higher R1 and lower R2 relaxation rates, which is characteristic of a greater mobility in that portion of the polypeptide chain. For apoE-(126–183), no such regions are observed in the measured relaxation data. In general, relaxation rates are field-dependent: as magnetic field strength increases, R1 decreases, whereas the {1H}-15N heteronuclear NOE and to a much lesser extent R2 increase. The only parameter sensitive to millisecond to microsecond time scale motions is R2. Large differences in R2 at different field strengths imply that motion is occurring in the millisecond to microsecond time scale. This motion is termed exchange and can arise from structural interconversions or aggregation. It is unlikely that apoE-(126–183) forms an oligomer as these studies were performed in 50% TFE. TFE has a tendency to weaken hydrophobic interactions and has been shown to be a denaturant of quaternary structure (41Slupsky C.M. Kay C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: 7365-7375Crossref PubMed Scopus (61) Google Scholar, 42Buck M. Q. Rev. Biophys. 1998; 31: 297-355Crossref PubMed Scopus (706) Google Scholar). Fig. 4shows typical behavior for a monomeric protein. R1 and NOE show the proper field dependence, whereas R2 shows very little field dependence, ruling out exchange as a mechanism for describing the motions present in apoE-(126–183). The bottom panel in Fig. 4 shows the R2/R1 ratio. In general, R2/R1 ratios are determined by the overall rotational correlation time, assuming the value of R2 is not dominated by exchange (43Kay L.E. Torschia D.A. Bax A. Biochemistry. 1989; 28: 8972-8979Crossref PubMed Scopus (1774) Google Scholar, 44Brutscher B. Brüschweiler R. Ernst R.R. Biochemistry. 1997; 36: 13043-13053Crossref PubMed Scopus (161) Google Scholar), and the position of the N-H bond vector relative to the diffusion tensor of the protein. ApoE-(126–183) exhibits much larger R2/R1 ratios for the central residues (residues 149–159) than for the termini. Because the lack of field dependence of R2 indicates there are no significant exchange contributions, anisotropic motion was investigated as a source for the large R2/R1 values. Assuming that the protein is an extended helix and can be represented as a prolate ellipsoid, the motion will be anisotropic, and the rotational correlation times along the long axis (∥) and short axis (⊥) will be different. Assuming a diffusion tensor ratio D∥:D⊥ ∼7, expected for an extended helix of 58 residues, values of 0.7 s−1 are calculated for R1, 25 s−1 for R2, and 0.77 for the NOE (45Perrin F. J. Phys. Radium. 1934; 5: 497-511Crossref Google Scholar, 46Perrin F. J. Phys. Radium. 1936; 7: 1-11Crossref Google Scholar, 47Tjandra N. Feller S.E. Pastor R.W. Bax A. J. Am. Chem. Soc. 1995; 117: 12562-12566Crossref Scopus (663) Google Scholar). For residues 149–159, the average R1 is 0.95 s−1, R2 is 25 s−1, and the heteronuclear NOE is 0.56. The similarity of calculatedversus experimental values suggests that the motion of apoE-(126–183) approximates that of a prolate ellipsoid, at least for the central residues. The decrease in R2/R1 ratios on either side of the central region (residues 149–159) of the peptide implies that the helix is “fraying” or “unfolding” as one approaches the N or C terminus. To determine the time scales of the motions involved, we undertook a reduced spectral density approach to study the motions along the helix. The spectral densities quantitate the contributions of motions at various frequencies obtainable from the NMR experiment. Fig.5 illustrates the reduced spectral density functions J(0), J(ωN), andJ(0.87ωH), which describe the motion of the H-N bonds, derived from the relaxation parameters R1, R2, and {1H}-15N NOE. The spectral density function at zero frequency, J(0), is sensitive to motions on all time scales (48Viles J.H. Donne D. Kroon G. Prusiner S.B. Cohen F.E. Dyson H.J. Wright P.E. Biochemistry. 2001; 40: 2743-2753Crossref PubMed Scopus (164) Google Scholar). The high frequency spectral density functions, J(ωN) andJ(0.87ωH), are sensitive to fast internal motions on the time scales of 1/ωN and 1/wH, respectively. Large J(0.87ωH) (greater than 15 ps/rad) indicate fast internal motions, whereas spectral densities less than 7.5 ps/rad indicate a lack of internal flexibility (48Viles J.H. Donne D. Kroon G. Prusiner S.B. Cohen F.E. Dyson H.J. Wright P.E. Biochemistry. 2001; 40: 2743-2753Crossref PubMed Scopus (164) Google Scholar). Fig. 5 indicates the relative inflexibility of the central region of the peptide, especially on the faster time scales. Residues 144–162 exhibit J(0.87ωH) lower than 7.5 ps/rad, whereas J(0) is consistently above 7 ns/rad for residues 149–162. Not surprisingly, the first four residues, 127–130, and the last sev" @default.
• W1976501903 created "2016-06-24" @default.
• W1976501903 creator A5047794053 @default.
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• W1976501903 creator A5079084423 @default.
• W1976501903 date "2002-08-01" @default.
• W1976501903 modified "2023-09-30" @default.
• W1976501903 title "NMR Structure and Dynamics of a Receptor-active Apolipoprotein E Peptide" @default.
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• W1976501903 doi "https://doi.org/10.1074/jbc.m204043200" @default.
• W1976501903 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12036962" @default.
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