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Originally published In Press as doi:10.1074/jbc.M608085200 on November 1, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39294-39299, December 22, 2006
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Lipid-induced Extension of Apolipoprotein E Helix 4 Correlates with Low Density Lipoprotein Receptor Binding Ability*

Vinita Gupta{ddagger}, Vasanthy Narayanaswami{ddagger}, Madhu S. Budamagunta§, Taichi Yamamato{ddagger}, John C. Voss§, and Robert O. Ryan{ddagger}1

From the {ddagger}Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, Oakland, California 94609 and §Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, California 95616

Received for publication, August 23, 2006 , and in revised form, October 31, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to {alpha} helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apolipoprotein E (ApoE)2 is an anti-atherogenic protein that regulates cholesterol levels in plasma by virtue of its ability to serve as a high affinity ligand for members of the low density lipoprotein receptor (LDLR) family (1, 2). Transgenic mice deficient in apoE display severe hypercholesterolemia and develop spontaneous and diet-induced atherosclerosis (3, 4). Conversely, mice overexpressing apoE display a marked regression of atherosclerotic lesions and resistance to diet-induced atherosclerosis (5, 6). ApoE occurs as three different isoforms, apoE2, apoE3, and apoE4, which differ in residues at positions 112 and 158. ApoE3, the most common isoform, bears a Cys and Arg at these positions, respectively.

ApoE is composed of two independently folded structural domains that are linked by a protease sensitive loop (7, 8). The 22-kDa N-terminal (NT) domain bears amino acids critical for LDLR binding, whereas the 10-kDa C-terminal domain manifests high lipid binding affinity and is responsible for apoE self-association in the absence of lipid. X-ray crystallography of apoE3-NT reveals a 4-helix bundle molecular architecture (9). The classical LDLR recognition sequence encompasses a cluster of positively charged amino acids in the region of residues 134-150 on helix 4 of the N-terminal domain (7). Another positively charged residue, Arg-72, has also been implicated in LDLR binding (8). In addition, lipid association appears to be an essential prerequisite for apoE binding to LDLR. Interestingly, full-length apoE and the NT domain are equivalent in terms of LDLR binding ability (7). Reconstituted apoE (full-length or NT domain) phospholipid disks possess full receptor binding activity and provide a convenient model for studies of the lipid-associated conformation of apoE. Infrared spectroscopy studies show that helical segments in apoE3-NT domain align perpendicular to phospholipid fatty acyl chains around the perimeter of bilayer disk complexes (10). In fluorescence resonance energy transfer studies it has been reported that apoE3-NT adopts an extended conformation wherein the molecule aligns in tandem with a second apoE3-NT around the perimeter of the disk (11, 12). Spin labeled fatty acids and single tryptophan variants were used to show that apoE3-NT and full-length apoE adopt similar extended conformations (13).

In another approach, a peptide corresponding to apoE residues 126-183 was generated that possessed lipid and LDLR binding activity (14). Multidimensional NMR studies of apoE (126-183) associated with dodecylphosphocholine micelles revealed that residues known to exist as random coil in the lipid-free state adopt helical structure in the presence of lipid (15). Questions emerging from these studies relate to whether the lipid-induced random coil to {alpha}-helix transition of this 58-residue peptide fragment also applies to apoE-NT or full-length apoE. To address this question we employed site-directed spin label-electron paramagnetic resonance (SDSL-EPR) spectroscopy to investigate the effect of lipid interaction on spin label probes positioned at regular intervals between amino acids 161 and 181 in apoE-NT (see Fig. 1) and full-length apoE. The data reveal that lipid association induces residues beyond Ala-164 (the termination site of helix 4 in lipid-free apoE) to transition from random coil to a more stable backbone. We conclude that the molecular basis for the divergent behavior of lipid-free and lipid-bound apoE in terms of LDLR binding is related to lipid binding-induced extension of helical structure beyond its termination site in the helix bundle state.


Figure 1
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  		FIGURE 1.
Ribbon diagram of apoE3-NT helix bundle structure. The protein is depicted in its 4-helix bundle (green) conformation. The classical LDLR recognition sequence is shown in pink. Positions of single cysteine substitution mutations introduced into C112S ApoE-NT are denoted by white circles labeled according to sequence position. The figure was prepared from Protein Data Bank coordinates 1lpe using the PYMOL program.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Production and Bacterial Expression of apoE Variants—Site-directed mutagenesis was performed using the QuikChange XL kit from Stratagene according to the manufacturer's instructions, and all mutations were verified by dideoxy automated DNA sequencing. ApoE3-NT and full-length variants were expressed in Escherichia coli BL21 cells transformed with an apoE-pET-20b plasmid with His tag and purified on a Hi-Trap chelating nickel column from GE Healthcare as described (16). Samples were electrophoresed on precast 4-20% acrylamide gradient SDS gels (Invitrogen) at 30-mA constant current and stained with Coomassie Blue.

Nitroxide-labeled apoE—Spin labeling was carried out as described earlier (17). Briefly, 5 mg of apoE in buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4) containing 3 M guanidine-HCl, and 0.25 mM dithiothreitol was loaded onto a Hi-Trap column. The column was then washed with 3 column volumes of buffer A containing 3 M guanidine-HCl and 100 µM Tris(2-carboxyethyl)phosphine hydrochloride followed by three column volumes of buffer A containing 3 M guanidine-HCl plus 160 µM methane thiosulfonate (MTS) and incubated for 45 min at 25 °C. The column was then washed with 3 column volumes of buffer A and protein eluted with buffer A containing 0.5 M imidazole. Eluted protein was dialyzed to remove imidazole and stored at 4 °C.

ApoE·phospholipid Complexes—Dimyristoyl-phosphatidylcholine (DMPC)-apoE disks were prepared as described earlier (18). Briefly, DMPC was dissolved in chloroform:methanol (3:1 v/v) and dried into a thin film in a glass tube. Following dispersion of the DMPC in 10 mM Tris-HCl, 150 mM NaCl, pH 7.2 (TBS), MTS-labeled apoE variants were added (2.5:1 DMPC/apoE weight ratio). This mixture was subjected to bath sonication until clear. Solubilized apoE·DMPC complexes were dialyzed against TBS and stored at 4 °C until use. Native PAGE of DMPC·apoE complexes was performed to evaluate the size of lipoprotein complexes. For UV circular dichroism spectroscopy, analysis of apoE-NT single cysteine variants was conducted on a Jasco-710 spectropolarimeter maintained at 24 °C. The results indicated that {alpha}-helix content was not adversely affected by probe attachment. Furthermore, with each variant examined, association with DMPC induced a 2-12% increase in {alpha}-helix content.

Electron Paramagnetic Resonance Measurements—EPR spectra were recorded at 22 °C in TBS buffer on a JEOL-X band spectrometer equipped with a loop gap resonator in a single scan over 100 G as described earlier (17). The microwave power was set at 2 milliwatts, and the modulation amplitude was set at 1 G. C112S apoE-NT served as a control for MTS labeling. EPR spectroscopy of this protein yielded no signal, confirming the specificity of label incorporation.

LDLR Binding Assay—The ability of variant apoE-NT to interact with recombinant human LDLR was evaluated as described by Yamamoto et al. (19). Briefly, C112S apoE-NT (3 µg protein (lipid free or DMPC bound)) was pre-incubated for 5 min with 4 µg of recombinant LDLR (residues 1-699) followed by the addition of 1 µg of tryptophan-null apoE-NT·DMPC that was labeled on Cys-112 with the fluorescent probe, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS). The mixtures were incubated in 20 mM Tris, pH 7.2, 2 mM CaCl2, 90 mM NaCl at 25 °C for 1 h prior to fluorescence analysis. Fluorescence measurements were performed at 25 °C on a Perkin-Elmer Life Sciences LS50B luminescence spectrometer as described (19). In other experiments, specified single Cys substitution variants of C112S apoE-NT were labeled with AEDANS, and fluorescence emission spectra of the labeled variants (~5 µg of protein) were recorded in lipid-free or lipid-bound state as described earlier (20, 12). The excitation wavelength was set at 345 nm and the fluorescence emission spectra acquired between 350 and 600 nm, with a slit width of 3.5 nm.


Figure 2
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  		FIGURE 2.
Binding of variant apoE-NT to LDLR. One µg of AEDANS labeled tryptophan null apoE3-NT DMPC and 4 µg of a soluble fragment of LDLR (residues 1-699) were incubated in the absence or presence of 3-µg unlabeled lipid-free C112S apoE-NT or C112S-apoE-NT DMPC. Samples (300-µl final volume) were excited at 280 nm and fluorescence emission intensity at 470 nm determined. Values reported are the average ± S.D. (n = 3) of the percentage maximal fluorescence enhancement at 470 nm (excitation 280 nm) observed in the absence of competitor ligand.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Study Design and Rationale—To investigate the hypothesis that lipid association induces residues beyond the termination site in helix 4 to adopt a helical conformation that confers LDLR recognition properties, SDSL-EPR studies were designed. Initially, Cys-112 in apoE3-NT was substituted with serine. This conservative substitution was necessary to permit subsequent specific labeling of the engineered cysteines. Prior to introducing new cysteine mutations, C112S apoE-NT was expressed and characterized in terms of secondary structure and its LDLR binding activity. Far UV circular dichroism spectroscopy showed that the secondary structure content of the protein was unaffected by the C112S mutation. Furthermore, expected increases (13) in negative ellipticity were noted for spectra obtained in the presence of the helix inducing co-solvent trifluoroethanol (50% v/v) or when present as DMPC disks (data not shown). The results of LDLR binding assays (Fig. 2) revealed that, whereas lipid-free C112S apoE-NT is a poor competitor ligand, upon association with DMPC, this variant manifests LDLR binding activity equivalent to wild type (WT) apoE3-NT. Based on these results, C112S apoE-NT was used as a template to generate a series of single cysteine substitution apoE variants that were then labeled with MTS and subjected to EPR spectroscopy in their lipid-free and lipid-bound states.


Figure 3
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  		FIGURE 3.
SDS-PAGE of apoE-NT variants. Protein samples were electrophoresed under (a) reducing and (b) nonreducing conditions. Lane 1, molecular weight markers; lane 2, WT apoE3-NT; lane 3, C112S apoE3-NT; lane 4, G169C apoE-NT; lane 5, G173C apoE-NT; lane 6, A176C apoE-NT; and lane 7, L181C apoE-NT. ApoE-NT samples in lanes 4-7 also harbored the C112S substitution. The gels were stained with Coomassie Blue.

 


Figure 4
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  		FIGURE 4.
Native PAGE of apoE-NT·DMPC complexes. DMPC complexes were prepared with MTS-labeled apoE-NT variants and electrophoresed for 24 h under native conditions at 125 V. Lane 1, molecular weight markers; lane 2, C112S apoE-NT disks; lane 3, C112S/G169C apoE-NT disks; lane 4, C112S/G173C apoE-NT disks; lane 5, C112S/A176C apoE-NT disks; and lane 6, C112S/L181C apoE-NT disks.

 
Characterization of Single Cysteine Variants—Single cysteine apoE-NT variants, including V161C, G165C, G169C, G173C, A176C, and L181C, were analyzed by SDS-PAGE. The gel revealed a single band whose migration properties were indistinguishable from that of WT apoE3-NT (Fig. 3a). A corresponding gel electrophoresed under nonreducing conditions (Fig. 3b) revealed the presence of significant amounts of disulfide-linked homodimers in WT apoE3-NT and the different cysteine substitution variants. As expected, C112S apoE-NT did not show evidence of dimer formation. The dimer band present in each of the cysteine substitution mutants migrated faster than WT apoE3-NT dimer. This may be related to the fact that Cys-112 was located in a more central region of the 4-helix bundle, and upon disulfide formation, dimers were less able to fully extend in the presence of SDS compared with variants with a cysteine in closer proximity to the C terminus of the protein. Cysteine residues in different apoE-NT variants were then labeled with MTS, reconstituted into DMPC disks, and characterized by nondenaturing pore limiting gradient PAGE (Fig. 4). The gel indicates that C112S apoE-NT and MTS-labeled apoE-NT variants associated with DMPC to form lipid complexes of similar size (~17 nm). ApoE3-NT-DMPC particles of this size were previously reported to possess between 4 and 6 apoE molecules per disk complex (12).


Figure 5
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  		FIGURE 5.
Comparison of EPR spectra of lipid-free and lipid-bound apoE-NT variants. EPR spectra of MTS labeled apoE-NT variants in lipid-free (black) and DMPC-bound (red) states were recorded in Tris-buffered saline using 30 µg of labeled protein at a concentration of 4 mg/ml. The spectra were normalized with respect to spins/mg protein for each sample.

 
EPR Analysis of MTS-labeled apoE-NT Variants—EPR spectroscopy was performed on MTS-apoE-NT variants (Fig. 5). Representative EPR spectra of lipid-free (black) and lipid-bound (red) apoE-NT variants spin-labeled at positions 161, 165, 169, 173, 176, and 181 are shown. In the lipid-free state, apoE-NT-bearing spin labels at positions 173, 176, and 181 display similar narrow-lined spectra. This is typical of spin labels undergoing rapid tumbling motion and is indicative of a dynamic and unstructured state for the backbone of these residues. In contrast, the EPR spectrum of lipid-free apoE-NT spin labeled at position 161 displays a broadened spectrum, indicative of a site located along a region of fixed secondary structure. Introducing a spin label at this position served as a positive control because x-ray crystallographic analysis indicates that residue 161 forms part of helix 4, which encompasses residues 130-164 (9). The spectrum obtained for this variant, which reveals an immobilized residue in a surface helical location, establishes the validity of this approach (21). ApoE-NT spin-labeled at position 165 displays a similar line shape, suggesting a relatively structured helical state for this site. The spectrum of apoE-NT spin-labeled at position 169 reveals a small extent of narrowing of spectral line shape, suggesting a more flexible, unstructured state, intermediate between that observed for residues 165 and 173.

Upon lipid interaction, there was a dramatic alteration in the EPR spectral line shapes of all variants, which was particularly evident with labels at positions 173, 176, and 181. The observed line broadening is a characteristic feature of immobilized structures, consistent with stable interaction of these sites with the lipid substrate. Interestingly, the broad splitting in the spectrum of apoE-NT labeled at position 176 is indicative of a strongly immobilized side chain, suggesting this position may represent a point of stable tertiary or quaternary contact in the lipid-bound state.

Fluorescence Studies of apoE-NT Variants—To obtain independent verification that labeled residues transitioned to a more hydrophobic environment when the molecule was complexed with DMPC, fluorescence studies were performed. ApoE-NT single cysteine variants were labeled with the environmentally sensitive fluorescence probe, AEDANS (22), and emission spectra of lipid-free and lipid-bound proteins recorded. With the exception of position 161, lipid association induced a blue shift in wavelength of maximal fluorescence emission ({lambda}max) compared with corresponding spectra obtained in the lipid-free state (Table 1). The observed 14-nm red shift, upon lipid association, is consistent with data on the apoE (126-183) peptide. In the lipid-associated state, this residue was located on the polar face of the amphipathic {alpha} helix. In the case of G165C and G169C apoE-NT, the shift was relatively small (~3 nm). On the other hand, the spectra of AEDANS-labeled A176C and L181C displayed a blue shift of 12-17 nm in the {lambda}max upon association with DMPC. Thus, modified residues that adopted helical secondary structure upon lipid association also transitioned to a more hydrophobic environment.


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  		TABLE 1
Fluorescence properties of AEDANS-labeled apoE-NT variants

 


Figure 6
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  		FIGURE 6.
EPR spectra of full-length apoE variants. EPR spectra of MTS-labeled apoE variants in lipid-free (black) and DMPC-bound (red) states were recorded in Tris-buffered saline using 30 µg of labeled protein at a concentration of 2 mg/ml. The spectra were normalized with respect to spins/mg protein for each sample.

 
EPR Analysis of Full-length apoE Variants—A question emerging from studies with the NT domain of apoE is whether results obtained can be extended to the full-length protein. To address this, full-length C112S apoE containing the same single Cys substitutions were spin-labeled and analyzed by EPR (Fig. 6). For each of the labeled variants examined, the spectral line shapes observed in buffer, as well as their response to lipid interaction, were similar to those observed for apoE-NT variants. These data suggest that secondary structure (or lack thereof) in this region of the protein is not substantially influenced by the C-terminal domain, consistent with the concept of independently folded N- and C-terminal domains initially proposed by Weisgraber and coworkers (23, 24).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The function of apoE as a regulator of plasma cholesterol has been attributed to its ability to bind LDLR family members. Previous studies have mapped the classical LDLR binding site of apoE to the region encompassing residues 134-150 (7). Using various deletion mutants, it has been shown that residues 171-183 are also critical for manifestation of the LDLR binding activity of apoE (25). Subsequently, Morrow et al. (8) determined that mutating Arg-172 results in loss of LDLR binding activity. Because lipid-free apoE also lacks LDLR binding activity, it is presumed that lipid association induces a change in the alignment or secondary structure of this residue such that it adopts a configuration appropriate for receptor interaction. The concept that correct presentation of positively charged amino acid side chains of apoE to ligand binding repeats of the LDLR is supported by the recent results (26). By x-ray crystallography, it has been demonstrated that binding of an alternative ligand, the receptor associated protein to a two module segment of LDLR involves two docking sites wherein conserved, calcium coordinated acidic residues interact with positively charged amino acids in the receptor associated protein. In addition, binding avidity was shown to be regulated by interaction of ligands with more than one binding module, suggesting a general mechanism for binding of other basic ligands to LDLR family members. The present results are consistent with the concept that lipid association-induced extension of helix 4 modulates receptor binding avidity by facilitating interaction of the region of apoE encompassing residues 130-180 with multiple ligand binding modules.

These observations are also in keeping with studies of a 58-residue apoE-derived peptide that encompasses apoE amino acids 126-183. Although this peptide is unstructured in solution, it adopts an extended {alpha}-helix conformation in the presence of the lipid mimetic co-solvent, trifluoroethanol (27) or when complexed to dodecylphosphocholine micelles (15). At the same time, however, an alternative model of lipid-bound apoE has emerged recently (28). On the basis of x-ray crystallography studies of apoE·dipalmitoylphosphatidylcholine complexes, it was proposed that residues between 160 and 170 formed a hairpin loop that allowed for alignment of Arg-172 with the main receptor binding site (residues 142-149). In this manner, it is conceivable that a single apoE molecule could interact optimally with one LDLR ligand binding module. Whereas EPR spectra of apoE variants harboring spin labels at positions 161, 165, and 169 are consistent with an extended structure such as that reported earlier (13), the model reported in Ref. 28 is also compatible with the data. In comparing models, however, it is important to consider differences in the respective experimental systems. For example, the present study was conducted using bilayer disk complexes of DMPC and apoE, whereas Peters-Libeu et al. (28) studied spheroidal complexes generated using dipalmitoylphosphatidylcholine. Furthermore, in contrast to the present study, which employed an apoE3-like isoform, Peters-Libeu et al. (27) used the apoE4 isoform. Given that the structure and function of this isoform is profoundly influenced by N- and C-terminal domain interactions (29-31), it is conceivable that apoE4 adopts an isoform-specific lipid-bound conformation. Further studies are required to explain isoform-specific differences in lipid-associated apoE.

It is well known that residues beyond amino acid 164 are poorly defined in the electron density map of lipid-free apoE3-NT (9). Our SDSL-EPR studies, however, provide evidence that amino acid residues at positions 165 and 169 retain some helical character in buffer alone. Beyond position 169, however, apoE exists as random structure in solution. Upon lipidation significant increases in the {alpha}-helix structure at each of the sites examined was observed, as illustrated by the substantial EPR spectral line shape changes. Thus, these sites and the region of 173-181, in particular, represent useful markers for lipid-binding events associated with apoE function. Although the precise structural organization of lipid-associated, LDLR active apoE is not known, available data are consistent with the following concept: lipid-free apoE is attracted to the surface of circulating lipoproteins by the high lipid binding affinity of the C-terminal domain. Once bound to the lipoprotein particle surface, the NT helix bundle can either retain its receptor inactive helix bundle conformation or undergo a conformational change (32-34) that results in stable contact with the lipid surface and manifestation of LDLR binding. It is possible that the NT domain reversibly transitions between an open, receptor-active conformation and a receptor-inactive helix bundle state although anchored to the lipoprotein surface via its C-terminal domain. Such a scenario suggests a molecular mechanism for regulation of lipoprotein clearance based on the conformational status of the NT domain.

A key question remaining relates to why, in the absence of lipid, apoE-NT domain adopts a 4-helix bundle in which helix 4 terminates at residue 164. Although is seems very likely that folding of the domain into a globular 4-helix bundle affords considerable stability via hydrophobic helix-helix interactions, it has also been reported that the region of apoE around residue 165 conforms to a classical Schellman helix cap motif (35, 15) commonly found at the end of {alpha} helices. If the termination of helix 4 in apoE is the result of a helix cap motif, then it is possible that lipid interaction induces a disruption of the cap motif, promoting extension of the helix and subsequent alignment of key positively charged residues for optimal interaction with ligand binding modules of the LDLR.


   FOOTNOTES
 
* This work was supported by the National Institutes of Health Grant (HL-64159). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Inst., 5700 Martin Luther King Jr. Way, Oakland, CA 94609. Tel.: 510-450-7645; Fax: 510-450-7910; E-mail: rryan{at}chori.org.

2 The abbreviations used are: apoE, apolipoprotein E; LDLR, low density lipoprotein receptor; NT, N-terminal; SDSL, site-directed spin label; EPR, electron paramagnetic resonance; MTS, methane thiosulfonate; DMPC, dimyristoyl-phosphatidylcholine; TBS, Tris-buffered saline; AEDANS, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Susan Marqusee, University of California, Berkeley, for providing access to the circular dichroism spectrometer; Dr. Paul M. M. Weers for assistance with spectroscopic analysis; and Lawrence Wang for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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