Note: Descriptions are shown in the official language in which they were submitted.
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1
AGONIST VERSUS ANTAGONIST BINDING
TO G PROTEIN-COUPLED RECEPTORS
U.S. GOVERNMENT RIGHTS
This invention was supported in part by grants from the
National Science Foundation (MCB-9904753), and the U.S.
Public Health Service, National Institute of Drug Abuse
(DA-06284). The Government has certain rights in the
invention.
CROSS-REFERENCE TO RELATED APPLICATION
This application is based on U.S. Provisional Application
No. 60/213,575, filed on 06/2/2000, and entitled "Plasmon
Resonance Studies of Agonist/Antagonist Binding to the
Human Delta-Opioid Receptor: New Structural Insights into
Receptor-Ligand Interactions."
BACKGROUND OF THE INVENTION
Field of the Invention
This invention pertains in general to the field of surface
plasmon resonance (SPR) spectroscopy. In particular, the
invention relates to a novel SPR approach involving the
coupling of plasmon resonances in a thin metal film and
the waveguide modes in a dielectric overcoating and the
use of such coupled plasmon-waveguide resonance (CPWR)
spectroscopy to study structural changes accompanying the
binding of ligands to G protein-coupled receptors
immobilized in a solid-supported lipid bilayer. ,
Descrixation of the Related Art
Most ligands responsible for cell-cell signaling
(including neurotransmitters, peptide hormones, and growth
factors) bind to receptors on the surface of their target
cells. Thus, deciphering the mechanisms by which cell-
surface receptors and their ligands mediate signaling
remains an important focus of study in biology.
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The majority of transmembrane signal transduction
responses to hormones, neurotransmitters,,phospholipids,
photons, odorants, and growth factors are mediated by a
superfamily (containing nearly 2,000 members and growing)
of seven transmembrane helical G protein-coupled receptors
,(GPCR). Activation of these receptors is assumed to
require protein conformational changes, which are induced
by the binding of ligands, to the receptor'/.s ~extracellular
domain. Subsequently, an associated G protein separates
from the receptor and conveys a signal to an intracellular
target, such as an enzyme or ion channel.
Current methods used to examine ligand-bineling
interactions with GPCRs, as well as with other membrane-
bound receptors, suffer,from several deficiencies. These
include the use of radiolabeled ligands, which require
special synthetic methodologies and present special
disposal and potential toxicity problems., In some cases,
ligands with fluorescent probes can be used, but the
modification of the ligand by the fluorophore often leads
t;o changes in the binding ,and other physical/chemical
properties of the ligand. Perhaps most importantly,
current binding methods, whether using radiolabeled or
fluorescent-labeled ligands, provide no information
regarding the changes in receptor structure that accompany
ligand-receptor interactions, nor do they distinguish the
different structural changes that occur for agonists and
antagonists interacting with the same receptor.
Thus, there remains a need in the art for~new and improved
ways of characterizing the biophysical properties of
ligand-GPCR interactions., As described herein, a new
method employing plasmon resonance spectroscopy is
utilized to characterize the binding interactions of
peptide ligands with a GPCR. The information thereby
obtained includes the direct determination,of the
thermodynamic binding constant for the non-covalent
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ligand-receptor interaction, and an assessment of the
structural changes that accompany this interaction, all in
a single highly sensitive measurement using unmodified
materials.
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SUN~1ARY' OF THE INVENTION
The invention meets the aforementioned need by providing a
new and improved method of characterizing the biophysical
properties of GPCR-ligand interactions. In general, the
inventive method utilizes a newly developed variant of the
surface plasmon resonance~(SPR) technique,referred to as
coupled plasmon-waveguide resonance (CPWR)j spectroscopy
(Salamon et al., Biophys. J. 73, 2791-2797, 1997; Salamon
et al. Trends Biochem. Sci. 24:213-219, 1999; Salamon and
Tollin, Encyclopedia of Spectroscopy and Spectrometry,
Vol. 3 J.C. Lindon, et al. Eds. Academic Press, San
Diego, pp. 2311-2319, 1999; Encyclopedia ,of Analytical
Chemistry R.A. Meyers, Ed. Wiley, New York, 2000; See
also U.S. Patents 5,521,702 and 5,991,488~both issued to
Salamon et al.) that allows the characterization of
anisotropic membrane systems (Salamon et al., Biophys. J.
73, 2791-2797, 1997; Biophys. J. 75:1874-1885, 1998;
Trends Biochem. Sci. 24:213-219, 1999), as well as other
anisotropic nanostructures (Salamon and Tollin,
Encyclopedia of Spectroscopy and Spectrometry, Vol. 3 J.C.
Lindon, et al. Eds. Academic Press, San Diego, pp. 2294-
2302, 1999; Encyclopedia of Analytical Chemistry R.A.
Meyers, Ed. Wiley, New York, 2000).
An object of the invention is to provide a method of
characterizing the biophysical properties of G protein-
coupled receptors, and their interactions with ligands,
that is more rapid and direct then existing methodologies.
A second object of the invention is to provide a highly
sensitive method of characterizing the biophysical
properties of G protein-coupled receptors and their
interactions with ligands.
A third object of the invention is to provide a method of
characterizing the biophysical properties of G protein-
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coupled receptors, and their interactions with ligands,
that does not produce toxic or radioactive waste products.
A fourth object of the invention is to provide a method of
5 characterizing the biophysical properties of G protein-
coupled receptors, and their interactions with ligands,
that does not modify the physical or chemical properties
of the molecules being characterized.
A fifth object is to provide a method of distinguishing
between agonist and antagonist ligands of G protein-
coupled receptors.
In accordance with these and other objects, the inventive
method has the unique capability of independently
examining real-time changes in the structure of a GPCR,
both parallel and perpendicular to the lipid membrane
plane, in response to receptor-ligand interactions. The
method also provides greatly enhanced sensitivity and
spectral resolution compared to conventional SPR. For
example, only femtomole amounts of receptor (and ligand)
are needed for complete spectral determination and
analysis. Furthermore, since radioactivity measurements
do not need to be performed, the methodology is much more
rapid and direct in the determination of critical binding
parameters. '
The invention thus provides a general procedure that can
replace previous methods in characterizing ligand-GPCR
interactions, and which at the same time can provide new
information about ligand-GPCR structural transitions that
are not available using prior methodologies.
The inventive method is illustrated herein via'
incorporation of the human b-opioid receptor into a pre-
formed lipid bilayer, examination of the binding to the
receptor of the highly.selective ligand DPDPE (c-[D-Pent,
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D-Pens] enkephalin; (Mosberg et al . , PNAS 80 : 5871-5874,
1983), demonstration of,the reversal of binding using the
selective antagonist naltrindol (NTI; Raynor et al., Mol.
Pharmacol. 45:330-334, 1994; Korlipara et,al., J. Med.
Chem. 38:1337-1343, 1995),, and evaluation. of the changes
in the receptor structure which accompany these binding
interactions. Significantly different structural changes
are shown to be induced in the S-opioid receptor upon
binding with either DPDPE or NTI, thereby providing new
insights into the structural basis of receptor function.
Various other purposes and advantages of the invention
will become clear from its description in, the
specification that follows,and from the novel features
particularly pointed out in the appended.claims.
Therefore, to the accomplishment of the objectives
described above, this invention consists of the features
hereinafter illustrated in the drawings, fully described
in the detailed description of the preferred embodiments,
and particularly pointed out in the claims. However, such
drawings and description disclose only some of the various
ways in which the invention may be practiced.
BRILF D$SCRIPTION OF THE DRAWINGS
Fig. 1: CPWR spectra obtained for a supported lipid
bilayer containing 75 mol% egg phosphatidylcholine and 25
mol% phosphatidylglycerol before (curve 1) and after the
addition of aliquots of human a-opioid receptor in octyl
glucoside-containing buffer into the aqueous compartment
of the CPWR cell (final receptor concentrationlin the bulk
solution for curve 2 is 4.8 nM and for curve 3 is 12.8
nM). The buffer composition was 10 mM Tris (pH 7.3), 0.5
mM EDTA and 10 mM KC1. The octyl glucoside concentration
in the receptor solution was 30 mM; after dilution into
the sample cell the concentration ranged from 0 to 5 mM.
Data obtained with p-polarized (panel A) and s-polarized
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(panel B) exciting light.are shown. Dotted lines
represent theoretical fits. The refractive index and
thickness values obtained from these fits are~given in
Fig. 4. In all cases, CPWR spectra were obtained after
equilibrium was reached (20-40 minutes).
.Figs. 2A and 2B: CPWR spectra obtained in an experiment in
which addition into the aqueous compartment of agonist
(DPDPE) is followed by addition of antagonist (NTI). The
cell contained a lipid membrane with the''receptor
incorporated (final bulk receptor concentration was 12.8
nM; spectra shown in curves 1 in panels A and B of Fig.
2A). Curves 2 in panels A and B show the. spectra obtained
after addition of 79 nM DPDPE. Curve 1 in panels C and D
(Fig. 2B) is the same as curve 2 in panels A and B. Curves
2 in panels C and D show the spectra obtained after
addition of 0.64 nM NTI. Other conditions as in Fig. I.
Fig. 3A and 3B: CPWR spectra obtained in experiment in
which addition of antagonist is followed by addition of
agonist. The cell contained a lipid membrane containing
the receptor, as in Fig. 2,(final bulk concentration was
12.8 nM; spectra shown in curves 1, panels A and B of Fig.
3A). Curves 2 in pan.els'A and B show the~spectra obtained
after addition of 0.144 nM NTI. This was followed by
addition of 360 nM DPDPE (curve 2; panels C and D of Fig.
3B). Curve 1 in panels C'and D is the same as curve 2 in
panels A and B. Other conditions as in Fig. 1.
Fig. 4: Dependence of the relative position of the CPWR
resonance minimum on the agonist (DPDPE) and antagonist
(NTI) concentrations in the sample compartment of the
cell, obtained using either p- (circles) or s-polarization
(triangles). Resonance position displacement towards
higher values represents shifts to larger,angles of
incidence. Results were obtained in a continuation of. the
experiment described in Fig. 2. After receptor
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8
incorporation, aliquots of the agonist solution in buffer
were added; this was followed by addition of aliquots of
the antagonist solution. Other conditions as in Fig. 1.
Figure 5: Dependence of: the relative position of the CPWR
resonance minimum on the antagonist (NTI) and agonist
(DPDPE) concentration in the sample compartment of the
cell, obtained using either p- (circles) or s-polarization
(triangles). Resonance position displacement towards
higher values represents,shifts to larger angles of
incidence. Results were obtained in a continuation of the
experiment described in Fig. 3. After receptor
incorporation, aliquots of antagonist solution in buffer
were added; this was followed by addition of agonist
solution. Conditions as in Fig. 1.
Figure 6: CPWR time-resolved spectra obtained with s-
polarized light, using a lipid membrane containing
receptor (final bulk concentration 12.8 nM) as described
in Fig. 2 (curve l). Curves 1 and 2 were obtained 20 s
and 60 s after addition of 7 nM agonist to the CPWR sample
cell, respectively. Insert shows the resonance position
shift as a function of time after addition of agonist.
Figure 7: Refractive index (panel A) and thickness (panel
B; triangles) values of the proteolipid film as a function
of the receptor concentration, obtained from theoretical
fits as described in Fig. 1. Data in panel A obtained
using either p- (circles) or s-polarized (triangles) light
(error bars due to uncertainties in curve fitting lie
within symbols). Panel B also shows the refractive index
anisotropy (A"; circles) as a function of the receptor
concentration. Solid lines in both panels represent
nonlinear least squares fits to a hyperbolic function.
These yield limiting values of both n (given in Figure)
and t (6.8 nm) extrapolated to infinite concentration of
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9
the receptor, as well as an apparent binding constant KD
(given in Figure).
Figure 8: Average change in thickness (circles) and
refractive index anisotropy values (triangles) for a
proteolipid membrane containing opioid receptor (bulk
concentration 12.8 nM) as a function of the agonist and
antagonist concentration. Results obtained from the
experiment in Fig. 4 by theoretical fits to the
experimental spectra (see Figs. 1 and 7). Insert shows
the square of the average refractive index of the
proteolipid membrane as a function of agonist
concentration. Solid lines represent nonlinear least
squares fits to a hyperbolic function from which the
binding constant values (KD) were obtained. For purposes
of clarity, error bars (corresponding to curve fitting
errors) for circles and triangles shown only for one of
the two curves in the main panel.
Figure 9: Average change in thickness (circles) and
refractive index anisotropy values (triangles) as a
function of antagonist,and agonist concentration. Results
obtained from the experiment in Fig. 5 by theoretical fits
to the experimental spectra. Other details as in Fig. 8.
Figure 10: Schematic representation of changes in
conformation (evaluated by refractive index anisotropy and
membrane thickness values)' and mass distribution
(evaluated by membrane thickness and average refractive
index values) of lipid and receptor molecules during
interaction of the receptor with either agonist or
antagonist molecules. For clarity, only four of the
transmembrane helices and the extramembrane loops of the
receptor are shown. Structural transitions~occurring upon
adding agonist subsequent to antagonist addition, and
antagonist subsequent to agonist addition are also shown.
See text for further description.
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DESCRIPTION OF THE PREFERRED ~ODIMBNTS OF THE INVENTION
The invention generally involves the application of
coupled plasmon wavelength resonance technology in a
5 method of characterizing the biophysical.properties of
membrane-bound G protein-coupled receptors. To illustrate
the invention, the human S-opioid~receptor was
incorporated into a pre-formed lipid bilayer and processed
as described hereinafter. However, this illustration~is
10 not intended to limit the method of,the invention to a
particular GPCR.
Purification of the Receptor ,
The human brain b-opioid;receptor, accession number U07882
(Knapp et al., Life Sci. 54:PL463-PL469, 1994), mediates
analgesic responses to endogenous enkephalins, as well as
to a variety of synthetic agonists. A fully functional
receptor, labeled at the C-terminus with a myc epitope
(Gimpl et al., Eur. J. Biochem. 237:768-777, 1996) and His
tag (Grisshammer and Tucker, Biochem. J.;317:891-899,
1996) was prepared by inserting the DNA of the human S-
opioid receptor, which was modified by incapacitating the
stop codon of the receptor, into the pcDNA3 vector
containing the myc/His tag (Invitrogen). The entire
vector was verified by DNA sequencing, and stably
transfected into a CHO cell line using DEAE-Dextran
(Promega). The transfected clones were selected using
6418 as an antibiotic. These were grown to confluency in
Ha mm's F12 medium with 10% fetal bovine serum containing
penicillin (100U/mL) and streptomycin (100 ~,g/mL) in a
humidified COZ atmosphere at 37°C. Related experiments
characterizing the modif~.ed receptor have been carried out
(See Okuara et al., Eur. J. Pharmacol. 387;RI1~R13, 2000).
After harvesting the cells and washing several times, they
were suspended in Tris-Cl buffer at pH=7.4 and centrifuged
at 42,000 rpm (160,000 X g) at 4°C~for 30 minutes. The
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buffer was decanted and the membranes were solubilized by
homogenization in a solution containing 25 mM Hepes, 0.5 M
KC1, 30 mM octylglucoside and protease inhibitors designed
to be used with metal chelating columns (Sigma), buffered
at pH = 7.4. After homogenization the solution was
centrifuged at 42,000 rpm again for 60 minutes to remove
cell debris.
The receptor was purified on a TALOI~1'rM Co+2 metal chelating
column (Clontech) with gentle rocking for 48 hours at 12°C
and eluted with 25 mM Hepes, 0.5 M KC1, 30 mM
octyglucoside and 100 mM imidazole buffered at pH = 7.4.
Although the. binding can be carried out in 24 hours, this
experiment was allowed to go for 48 hours in order to
maximize binding of the receptor to the TALON,column. The
column and receptor homogenate were kept at 12°C in order
to minimize any possible denaturation of the receptor due
to heat or protease, which may still be present in the
system. The concentration of receptor in the purified
sample was determined in a binding assay using'a
radioactive ligand (Okuara et al., Eur. J. Pharmacol.
387:811-813, 2000).
The agonist (DPDPE) used in this work was synthesized in
Dr . Victor Hruby' s laboratory (Mosberger et al' . , PNAS
80:5871-5874, 1983) and the antagonist (NTI) was obtained
from RBI Labs.
Formation of Solid-Supported Lipid Bilayers
Self-assembled solid-supported lipid membranes were
prepared according to the method used for formation of
freely suspended lipid bilayers (Mueller et al., Nature
194:979-980, 1962). This involves spreading a small amount
of lipid solution across an orifice in a Teflon sheet hat
separates the thin dielectric film (SiOZ) from the aqueous
phase (Salamon et al., Trends Biochem. Sci. 24:213-219,
1999; See also U.S. Patents 5,521,702 and 5,991,488 both
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issued to Salamon et al.). The hydrophilic surface of
hydrated Si02 attracts the polar groups of the lipid
molecules, thus inducing an initial orientation of the
lipid molecules, with the hydrocarbon chains pointing
toward the droplet of excess lipid solution. The next
steps of bilayer formation, induced by adding aqueous
buffer into the sample compartment of the CPWR cell,
involve a thinning process and formation of a plateau-
Gibbs border of lipid solution that anchors the membrane
to the Teflon spacer. In°the present experiments, the
lipid films were formed from a solution containing 5 mg/mL
egg phosphatidylcholine (PC) and 1-palmitoyl-2-oleoyl-sn-
glycero-3-[phospho-rac-(1-glycerol)(sodium salt) (POPG)
(75:25 mol/mol) in squalene/butanol/methanol
(0.05:9.5:0.5, v/v). The lipids were purchased, from
Avanti Polar Lipids Inc. (Birmingham, AL). All experiments
were carried out at ambient temperature using 10 mM Tris
buffer containing 0.5 mM EDTA and 10 mM KC1, pH 7,3, in
the 2 mL sample cell.
CPWR Sx~ectroscc~v
Details of the procedures for CPWR measurement and data
analysis have been described elsewhere (Salamon et al.,
Biophys. J. 73, 2791-2797, 1997; Trends Biochem. Sci.
24:213-219, 1999; Salamon;and Tollin, Encyclopedia of
Spectroscopy and Spectrometry, Vol. 3 J.C. Lindon, et al.
Eds. Academic Press, San Diego, pp. 2311-2319, 1999; See
also U.S. Patent 5,991,488 issued to Salamon et al.).
The method is based upon the resonant excitation by
polarized light from a CW He-Ne laser (~, = 632.8 nm),
passing through a glass prism under total internal
reflection conditions, of collective electronic
oscillations (plasmons) in a thin metal film (.Ag)
deposited on the external surface of the prism~which is
overcoated with a dielectric layer (Si02).
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13 , ,.
The resonant excitation of plasmons generates an
evanescent electromagnetic field localized at the outer
surface of the~dielectric film, which can be used to probe
the optical properties of molecules immobilized on this
surface (for further details see refs. Salamon et al.,
B.iophys. J. 73, 2791-2797,; 1997; Salamon a al. Trends
Biochem. Sci. 24:213-219, 1999; Salamon and Tollin,
Encyclopedia of Spectroscopy and Spectrometry, Vol. 3 J.C.
Lindon, et al. Eds. Academic Press, San Diego, pp. 2311-
2319, 1999; Encyclopedia of Analytical Chemistry R.A.
Meyers, Ed. Wiley, New York, 2000). Resonance is
achieved either by varying the incident-light wavelength
(,1~), at a fixed incident angle (a), or by varying a at a
fixed ~, (in the present experiments the latter protocol
was used). Because the resonance coupling generates
electromagnetic waves at .the expense of incident light
energy, the intensity of totally reflected light is
diminished. The reflected light intensity as a function
of either ~, or a results in a CPWR resonance spectrum..
The resonance can be excited with light polarized either
parallel (p) or perpendicular (s) to; the incident plane,
resulting in two well separated spectra (Salamon et al.,
Baophys. J. 73:2791-2797, 1997), thereby allowing
characterization of the molecular organization oft
anisotropic systems such as biomembranes containing
integral proteins (Salamon et al., Trends Biochem. Sci.
24:213-219, 1999. and references cited therein). Under
the experimental conditions employed in this work, the
optical parameters obtained with p-polarization refer to
the perpendicular direction, and with s-polarization to
the parallel direction, relative to the b~:layer membrane
surf ace .
CPWR spectra can be described by three parameters: a (or
~,); the spectral width; and the resonance depth. These
depend on the refractive index (n), the extinction
coefficient (k) and the thickness (t) of the plasmon-
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generating and emerging media, the latter including a thin
film deposited on the silica surface (i.e. a proteolipid
membrane in the present ease) in contact with an aqueous
solution. Thin-film electromagnetic theory based on
Maxwell's equations provides an analytical relationship
between the spectral parameters and the optical properties
of these media. This allows evaluation of n, k and t
uniquely for the three media (i.e., the plasmon-generating
medium, the proteolipid membrane and the aqueous buffer
solution), by non-linear least-squares fitting of the
theoretical spectrum to the experimental one (for details
see ref. Salamon et al.;Trends Biochem. Sci. 24:213-219,
1999; Biophys. J. 78:1400=1412, 2000; Salamon and Tollin,
Encyclopedia of Spectroscopy and Spectrometry, vol. 3.
J.C. Lindon et al. Eds.Academic Press, San Diego, pp.
2294-2302, 1999). Inasmuch as the excitation wavelength
(632.8 nm) is far removed from the absorption bands of the
lipids, protein and ligands used in this work, a k value
other than zero reflects a decrease of reflected light
intensity due only to scattering resulting from
imperfections in the proteolipid film.
It is important to point out that for an anisotropic thin
film, such as the proteolipid membrane in the present "
work, the thickness (t) represents an average molecular
length perpendicular to the plane of-the film, and will be
,.
independent of light polarization. In contrast, the
values of the refractive index (n) will be very much
dependent on the polarization of the excitation light.
Furthermore, for uniaxial~anisotropic structures in which
the optical axis is parallel to the p-polarization
direction, the n~ value will always be larger than nB.
This is a consequence of the fact that the measured
refractive index of a material is determined by the
polarizability of the individual molecules. The latter
property describes the ability of a molecule to interact
with an external electromagnetic field, and in general is
CA 02413081 2002-12-16
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anisotropic with respect to the molecular frame. In the
simplified case in which the molecular shape is rod-like
(e.g., the phospholipid molecules used in this work), one
can assign two different values to the polarizability: the
5 larger one, longitudinal and the smaller one, transverse.
If in addition to the anisotropy in molecular shape and
polarizability, the system which contains these molecules
10 is ordered such that the long axis of the molecules are
parallel, this results in long range order usually
described by the order parameter S. In this situation the
values of the polarizability, averaged over the, whole
system and measured either parallel or perpendicular to
15 the direction of the long axis of the molecules, will be
different (i.e., the parallel value will be larger than
the perpendicular one). These conditions create an
optically anisotropic system, with the optical axis
perpendicular to the plane of the proteolipid membrane,
and the values of the refractive index measured with two
polarizations of light (i . a . parallel, .ng, and
perpendicular, n8 to the, optical axis) will describe this
optical anisotropy (A") as~ follows: .
~ _ (n2p - n~A ) / (nZav + 2 )
(1)
In this equation n", is the average value of the refractive
index, and for a uniaxial system is given by:
" = 1/3 (nZp + 2n~g)
(2)
In summary, the anisotropy in the refractive index
reflects both the anisotropy in the molecular
polarizability and the degree of long range order of
molecules within the system, and therefore can be used as
a tool to analyze structural organization (i.e., molecular
orientation). This is particularly important in the
context of the present work in which structural
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16
alterations of a proteolipid membrane, consisting of a
single lipid bilayer with inserted receptor molecules,
caused by ligand binding have been monitored by changes in
the refractive index anisotropy.
Furthermore, as can be seen from the Lorentz-Lorenz
relation, the average value of the refractive index is
also directly related to the mass density (Born and Wolf,
Principles of Optics, Permamom Press, New York,,1965;
Cuypers et al., J. Biol. ehem. 258:2426-2431, 1983).
Thus, from the thickness of the proteolipid film and the
average value of the refractive index one can calculate
the surface mass density (or molecular packing density),
i.e., mass per unit surface area (or number of,moles per
unit surface area.
zn the present experiments, the plasmon-generating device
was calibrated by measuring the CPWR spectra obtained from
a bare silica surface in contact with aqueous buffer with
both p- and s-polarized light, and then fitting these with
theoretical curves. The goal of such a calibration is to
obtain the optical parameters of the silica layer (i.e.,
refractive indices, extinction coefficients and thickness)
used in these experiments. This provides an input set~of
data used in analyzing the resonance spectra obtained with
proteolipid membranes deposited on the silica surface.
Thus, the resonance spectra obtained after a single lipid
bilayer membrane was created on the hydrophilic surface of
silica were fit using these data,, yielding the optical
parameters (nE, n8, and t) for the lipid bilayer. These
allowed the calculation of the refractive index anisotropy
and the surface mass density (i.e., molecular packing
density) of the bilayer. After incorporation of the
receptor molecules into the lipid membrane, the resulting
CPWR spectra allowed the characterization the, structural,
consequences of receptor incorporation. Finally, addition
into the aqueous sample compartment of the CPWR cell of
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I7
v
either agonist or antagonist again resulted in changes of
the CPWR spectra, which reflected structural alterations
in the proteolipid membrane caused by the, receptor-ligand
interaction.
Incorporation ~f the b-Ox~ioid Receptor into a Pre formed
Lipid Bilayer
Receptor molecules were incorporated into a preformed ,
lipid membrane deposited on the hydrophilic surface of the
silica film by adding small aliquots of a concentrated
solution of the human b-opioid receptor solubilized in 30
inM octyl glucoside to the aqueous compartment of the CPWR
cell, thereby diluting the detergent to a final
concentration below its critical micelle concentration (25
mM) (Salamon et al., Biochemistry 33:13706-13711, 1994;
Biophy. J. 71:283-294, 1996). This resulted in
spontaneous transfer of the receptor from the micelle to
the lipid bilayer. The overall orientation of the
receptor in the bilayer~is not known. However,.as will be
shown below, ligand binding to the incorporated receptor
occurs efficiently, so that one can presume that at least
500 of the receptors are; bound with the liganding site
facing the aqueous buffer. .
Fig. 1 shows typical CPWR spectra, obtained with either p-
(panel A) or s-polarized exciting light (panel B), for a
solid-supported lipid membrane prior to (curve 1), and
after two additions of detergent-solubilized receptor to
the aqueous compartment of the sample cell.(cui~res 2 and
3). As noted previously with other integral membrane
proteins including rhodopsin (Salamon et al., Biochemistry
33:13706-13711, 1994; Biophys. J. 71:283-294, 1996),
protein incorporation into the bilayer influences all
three parameters of the resonance spectrum, i.e., angular
position, depth and spectral half-width. Such changes are
due both to mass density changes and to structural
alterations of the proteolipid membrane (reflected in
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18
I
changes in refractive index and thickness). These will be
considered further below.
Binding of Agonist (DPDPE) and Antaaonist (NTI) to
Incorporated Rece~,tor
This section describes the primary spectral data~obtained
upon adding DPDPE and NTI to the previously incorporated
receptor. As will be demonstrated, these data clearly
reveal different patterns of receptor-agonist and
receptor-antagonist interact~.on, thereby providing a
method of distinguishing agonist and antagonist binding.
When aliquots of either DPDPE or NTI solutions are added
to the sample cell after receptor incorporation into a
pre-formed bilayer, significant changes in the position,
width and depth of the CPWR resonance curve occur. These
spectral changes reflect the binding of these molecules to
the proteolipid membrane., Control experiments involving
addition of comparable amounts of these ligands to a CPWR
cell containing a preformed bilayer in the absence of
receptor produced no measurable effects on the CPWR
spectra (data not shown),, indicating that. non-specific
binding,to the membrane is not detected in these
experiments. Thus, the spectral changes observed when the
receptor is present must reflect receptor-ligand
interactions.
In order to illustrate these changes, examples of
resonance spectra obtained with both p- and s-polarized
exciting light are shown~in Figs. 2A and 2B and 3A and 3B.
Figs. 2A and 2B show the results of an experiment in
which agonist is added to the receptor-containing CPWR
cell first, followed by antagonist addition, and Figs. 3A
and 3B an experiment in which antagonist is added first
followed by agonist. As can clearly be seen, the effects
of these two ligands on the resonance spectra are easily
measurable and quite different. Although all three
CA 02413081 2002-12-16
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19
spectral parameters (i.:e.,,position,';width and depth) are
significantly altered by both ligands, appreciable
differences are seen in,the amplitude and~;direction of the
resonance shifts. Thus, DPDPE causes much larger changes
in both p- and s-polarized spectra (compare panels A and B
of Fig. 2A with panels A,and B of Fig. 3A) than are
induced by NTI. In addition, DPDPE shifts both resonances
to larger incident angle. values (see Figs. 2A and 3B),
although the change in the p-polarized signal is quite
small (see Fig. 5). In contrast, NTI moves the p-
polarized resonance to larger (see panel ;C of,Fig. 2B and
panel A of Fig. 3A), and the s-polarized ,resonance to
smaller, incident angles ,(see Figs. 2B and 3A).
To further illustrate these differences, plots ~of the
resonance position shifts as a function o.f added
concentration of the two~ligands are~shown in Figs. 4 and
5. These data also illustrate the fact that adding
antagonist after agonist does not simply reverse the
changes generated by agonist binding (see Fig.~4). In
contrast, agonist added after antagonist is bound is able
to reverse the changes caused by the receptor-antagonist
interaction (as can be clearly seen in the s-polarized
component in Fig. 5).
It is essential to emphasize that these spectral changes
saturate within concentration ranges (0-40, nM for DPDPE;
see Fig. 4, and 0-0.1 nM for NTI; see Figs 5) that are
consistent with literature data f.or the binding
characteristics of the ligands (see discussion below).
Thus, it is very unlikely that such high binding
affinities result from non-specific receptor-ligand
interactions. Furthermore, the results presented in Figs.
4 and 5 also clearly indicate that, although the direction
of the shifts remains the same regardless of which ligand
is added first, the concentration ranges in which the
resonance shifts occur depend on the sequence of addition
CA 02413081 2002-12-16
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r
(compare Figs. 4 and 5). Thus, in the experiments in
which agonist is added first, the antagonist concentration
range is significantly higher than that for the opposite
case. The same observation applies to the agonist when
5 the antagonist is added first.
Preliminary time-resolved measurements of; the CPWR spectra
following ligand addition demonstrate quite different
kinetic properties depending on which ligand is
10 interacting with the receptor. Fig. 6 shows an example of
such a time-dependent spectral sequence obtained with
DPDPE using s-polarized light. There are ,two significant
features o~ these spectral changes which distinguish the
receptor-agonist interaction from that of the'
15 receptor-antagonist interaction. First, agonist addition
results in a very slow (on the order of minutes) time
course of spectral changes, whereas antagonist addition
results in spectral changes that occur faster than the
resolution time (about 10s) of the present experiments.
Second, the kinetic properties of the spectral alterations
observed with the agonist are quite complicated, involving
negative shifts followed by positive shifts in an overall
multiphasic process (which we have not characterized in
full detail). Such results indicate a complex process of
receptor-ligand interaction. It is important to note that
a similar complex pattern of spectral changes is observed
with the p-polarized component (data not shown).
The above-noted differences between agonist and antagonist
binding properties cannot be explained simply by
differences in either the adsorbed mass of the ligand or
its rate of diffusion to the receptor, inasmuch as these
ligands have very comparable molecular masses (i.e., 648
for DPDPE and 414 for NTI). Furthermore, preliminary
experiments using another highly selective b-opioid
agonist, deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2;
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21
data not shown), reveal a similar kinetic pattern as
observed with DPDPE.
It is also important to note that the present data with
the b-opioid receptor have striking parallels to recent
studies with the i32 adrenergic receptor, in which
fluorescence spectroscopy was used to delineate. structural
changes associated with receptor-ligand interaction
,(Gether et al., FMF?O J. 16:6737-6747, 1997). In these
experiments the time course of fluorescence clearly
demonstrated that the kinetics of the receptor,-agonist
interaction are very comparable to those observed in the
present study (Fig. 6, insert), showing slow (on the order
of minutes) multiphasic kinetics, whereas the receptor-
antagonist interaction is much faster and,si.mpler.
Although it is clear that. further time-resolved studies
are necessary to fully understand the complexity of the
receptor-agonist interaction process, it is possible to
conclude from the present data that the interaction of the
b-opioid receptor with agonist or antagonist generates
different structural states of the proteolipid membrane,
whose properties depend on the sequence of ligand
addition. In order to provide a quantita ive description
of such states it is necessary to analyze the spectral
changes in more detail, taking into account alterations of
all three spectral parameters (i.e., resonance position,
depth and width). Such an analysis (see next section)
yields the optical parameters of the system, which can be
used in a quantitative characterization of the receptor-
ligand binding processes.
Characterization of the Receptor-cQn_tainina Livid Membrane
Quantitative analysis of the plasmon resonance spectra
obtained during receptor incorporation can be accomplished
by fitting theoretical curves to the experimental spectra
(see Fig. 1). Fig. 7 shows plots of~the optical.
CA 02413081 2002-12-16
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22
parameters obtained from ,such a procedure., (n [panel A] ; t
and An [panel B]; see Methods section for,parameter
definitions) as a function of added receptor. The solid
lines are single hyperbolic curves fitted to the data
points. These results indicate the following: 1) The
process of receptor incorporation is, satisfactorily fit by
a simple Langmuir isotherm; 2) The low value of the
apparent insertion constant (a 14 nM) argues for a quite
high efficiency of incorporation; 3) The extrapolated
thickness value (6.8 nm) describes the dimension of the
incorporated protein molecule perpendicular to the
membrane plane (i.e., the distance between the external
loops plus bound water on both sides of the membrane).
Extrapolation of the refractive index curves to infinite
receptor concentration (Fig. 7A) results in values (n~and
ng-) that characterize a monolayer of densely packed
receptor molecules. From these one can calculate an
average value of the refractive index (from equation [2]
above), and then mass density or surface concentration.
(Salamon et al., Trends B.iochem. Sci. 24:213-219, 1999).
From the latter value and the molecular weight~of the
receptor (using Mr= 60 kD), the surface area occupied by
one receptor molecule can be evaluated as.Sre~ = 1200~100
~ '
It is important to note that this value for the opioid
receptor is in very good agreement with reported values
for rhodopsin obtained with several techniques: SPR (1260
A2; Salamon et al., Biophys. J. 71:283-294, 1996), electron
cryomicroscopy (about 1000 A2; Schertler et al:, 1993;
Unger and Schertler, Biophys. J. 68:1776-1786, 1995), and
a rhodopsin Langmuir-Blodgett film by x-ray scattering
(about 1100 AZ; Maxia et al., Biophys. J. 69:1440-1446,
1995); 4) The increase in the proteolipid membrane
anisotropy occurring during the process of receptor
incorporation (shown in Fig. 7, panel B),,clearly reflects
a corresponding increase of the average long-range
CA 02413081 2002-12-16
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23
molecular order in the membrane resulting from receptor-
~.ipid interactions.
d
h r ri i n f R r-Li n In r i n
In general, CPWR spectral changes obtained with an
optically anisotropic thin proteolipid membrane (i.e.,
changes in position, depth and width) are the result of
both mass density (molecular packing density) and
structural alterations occurring within the system. The
mass density changes are directly reflected by the average
value of the refractive index changes (see equation [2]),
whereas structural alterations will influence the
refractive index anisotropy due to changes in 'the
orientational order of molecules within the membrane. The
latter quantity can be measured by changes in the
refractive index values obtained with p- and s-polarized
light (see equation [1], and further discussion below).
Distinguishing between these two types of changes is
especially important in the case of receptors, an which
the receptor-ligand interactions are thought to result in
structural alterations.
This distinction can be accomplished according to the
invention by fitting theoretical resonance curves to the
experimental CPWR spectra. Using the structural
parameters obtained for the receptor-containing
proteolipid membrane (described in the preceding section),
theoretical resonance spectra are fitted to the
experimental curves obtained in both agonist-antagonist
arid antagonist-agonist experiments (see Figs. 2 - 5). The
results, expressed as changes in refractive index
anisotropy, A", and proteolipid membrane thickness, as a
function of added Iigand are shown in Figs. 8 and 9,
respectively.
As noted above, binding of the two ligands drives the
proteolipid membrane system into distinct states
CA 02413081 2002-12-16
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r, ,
24
i ,
characterized by different spectral characteristics (see
Figs. 2 - 6). Based on the results in Figs. 8 and 9, one
can conclude that agonist binding (either before or after
antagonist binding) causes conformational changes in the
receptor molecule which result in net increases of both
anisotropy and mass density of the proteolipid system.
Mass density increases are shown by the increased values
of both na" (see insert in Fig. 8) and t (preliminary
experiments using another b-opioid receptor agonist,
deltorphin II, showed very similar changes in mass
density, A" and t upon binding to the receptor; data not
shown) .
~n contrast, antagonist binding to the receptor induces
only anisotropy changes, in the system (i.e., there are no
measurable changes in either na" or t values). These
conclusions are consistent with the data given in Figs. 2
- 5 and are especially well illustrated by the resonance
position shifts shown in,Fig. 4, in which the agonist
induces unidirectional (i.e. both p and s-components shift
in the same direction) whereas the antagonist induces
bidirectional resonance position shifts. Unidirectional
shifts of both spectral components in the agonist case is
clear evidence of an increase in both np and ne values
(i.e., an increase of the average refractive index value;
see equation [2]), which occurs as a result of a mass
density increase.
In contrast, addition of antagonist either before (Fig. 5)
or after (Fig. 4) agonist addition does not result in mass
density changes. In the latter case, all the spectral
changes axe related to structural alterations. Due to the
fact that both ligands have comparable molecular masses,
these results must be a consequence of addition of lipid
mass to the bilayer caused by the structural changes of
the receptor upon interaction with the agonist. (for
further discussion see below).
CA 02413081 2002-12-16
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It is also important to note that the conformational state
of the receptor induced by the antagonist has a much
higher refractive index anisotropy than that produced by
the agonist. This is clearly shown in both types of
5 experiments (see Figs. 8 and 9). Thus, when the agonist
is added before the antagonist, the latter ligand
increases the anisotropy to almost double the value
produced by the agonist. In contrast, when the'agonist is
added after the antagonist, the value of .A" is decreased to
10 a level comparable with the increase produced by the
agonist alone. In general, changes in refractive index
anisotropy are produced by alterations in the molecular
ordering with respect to the bilayer normal. In the
present invention, this must be a consequence of
15 conformational changes in the receptor molecules
accompanying ligand binding, i.e., changes in~position and
orientation of the transmembrane helices involving tilting
and rotational movements, as well as movements occurring
in the extramembrane loops. Changes in the aryl chain
20 ordering of the lipid molecules induced by these protein
structural alterations may also contribute.
In summary, the lack of measurable alterations in mass
density or membrane thickness upon antagonist binding
25 clearly implies a critical difference in the ',
conformational changes induced by such binding compared
with those induced by the agonist. This distinction is
also reflected in the fact that the state of the
proteolipid membrane created by the addition of antagonist
prior to agonist is different from that created when
antagonist is added after agonist addition. These
differences arise because the agonist is able to generate
structural alterations perpendicular to the plane of the
membrane changing its thickness, whereas the antagonist
cannot do so.
CA 02413081 2002-12-16
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26
Thus, the antagonist produces two sub-states depending
upon whether it is interacting with the unliganded
receptor, or with a receptor that has agonist bound to it
and therefore has changed its dimensions relative to the
membrane normal. Although these two sub-states are
characterized by similar optical anisotropies, they have
different dimensions and mass density. Since NTI is a
pure delta receptor antagonist with no reported partial
agonist biological activities (Wild et al., PNAS 91:12018-
12021, 1994) it is reasonable to conclude, that both of
these sub-states are inactive in signal transduction,
While it is possible that the short lived state represents
the receptor state that could lead to negative intrinsic
activity (Costa et al., Mol. Pharmacol. 41:290-297, 1992)
if it were long lived enough, a more likely possibility is
that the two sub-states represent non-equilibrium steady
states (Kenakin, Drugs 40:666-687, 1990) that are
accessible to the receptor, with one of these being only
transiently observed when NTI interacts with the delta,
opioid receptor. In order to obtain further insights into
these states, structural. changes in the lipid and protein
components must be separately determined for both agonist
and antagonist binding, and under a wide range of ratios
of agonist to antagonist. This can be done using
chromophore-labelled lipids, and such experiments are
underway.
Thermodynamic values for the individual ligand
dissociation constants can easily be evaluated from the
hyperbolic fits to the anisotropy changes presented in
Figs. 8 and 9. The results are given in Table 1. It is
evident that these dissociation constants strongly depend
on whether the agonist is present when the antagonist is
added, and vice versa. Thus, the presence of the other
ligand causes an appreciable shift of KD to higher values.
This observation is especially significant in the present
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27
system in which the antagonist has a much higher binding
affinity than the agonist (by 2-3 orders ~of magnitude).
Despite this, when NTI is"added after DPDPE, its
dissociation constant increases significantly~(about 4-
fold). This finding cannot be simply explained by
competition between these two ligands. This constitutes
another indication that different conformational states
are induced by these ligands, which are characterized by
different binding constants for the other.ligand.
I0
The binding constants determined here for;DPDPE and NTI
are similar to those reported in the literature using a
variety of b-opioid receptor membrane preparations and a
variety of radiolabeled competitive ligands. For DPDPE,
some typical values reported in the literature include
3.3-5.2 nM in several rat; brain membrane preparations
(Akiyama et al., PNAS 82:2543-2547, 1985), 1.2 nM for the
receptor cloned into the NG-108-15 cell line (Akiyama et
al., PNAS 82:2543-2547, 1985), and 85 nM for the receptor
cloned into the CHO cell, line (unpublished data). Thus,
the KD values of 10-40 nm reported here are consistent with
those expected for a fully functional receptor. Likewise,
the KD values previously reported (Wild, et al., PNAS
91:12018-12021, 1994) for NTI (0.9 nM in NG-108-15 cloned
receptors, 0.13 nM in mouse brain membranes and 0.15 nM in
mouse spinal chord preparations) are consistent~with the
values of 0.02-0.10 nM reported here.
Structural Basis of Recep~r Function
The CPWR results presented herein demonstrate the
formation of several conformational states of the
proteolipid membrane as a consequence of receptor-agonist
and receptor-antagonist interactions. In the case of
agonist binding, the slow multiphasic kinetics'clearly
indicate that there are a number of intermediate
conformational states involved in the formation of the
final activated state, as has been suggested by Gether°and
CA 02413081 2002-12-16
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28
Kobilka, J. Biol. Chem. 258:2426-2431, 1998. It is not
clear at present whether this final state involves an
equilibrium mixture of different conformational forms of
the receptor, or preferential formation of one particular
receptor structure (Kenakin, Trends Pharmacol. Sci.
16:232-238, 1995).
In either case, the present method has shown that the
receptor-agonist conformation produces an elongation of
the receptor molecule (increase in t), as well as an
overall increase in the degree of orientational order of
molecules within the membrane (increase in refractive
index anisotropy, An). It is reasonable to expect this
process to be relatively, slow because it also involves
alterations in the lipid phase of the membrane in response
to receptor elongation. Based on models for opioid
receptor structural changes upon activation (Pogozeva et
al., Biophys. J. 75:622-634, 1998; Knapp et al~.; FASEB J.
9:516-525, 1995; tether and Kobilka, J. Biol. Chem.
273:17979-17982, 1998) derived from studies of rhodopsin
(Farrens et al., Science 274:768-770, 1996),
bacteriorhodopsin (Luecke et al., Science;286:255-260,
1999), and the f~-adrenergic receptor (tether and Kobilka,
J. Biol. Chem. 273:17979-17982, 1998), we suggest that the
elongation process involves tilting and rotation of one or
more of the transmembrane helices resulting in vertical
movements of the extramembrane loops, and is accompanied
by movement of lipid molecules that cause an increase in
the positive curvature of the lipid surface.
The increase in curvature also requires the movement of
lipid molecules from the plateau-Gibbs border to the
bilayer phase, which increases the overall surface mass
density of the proteolipid membrane. The anisotropy
changes can be ascribed predominantly to orientation
changes of the transmembrane helices that influence the
ordering of the hydrocarbon chains of lipid molecules,
CA 02413081 2002-12-16
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29
without a significant contribution from the extracellular
loops or lipid mass redistribution. In contrast, the
binding of antagonist results only in an .increase in the
refractive index anisotropy, which implies localized
alterations occurring within the receptor molecule that
are restricted to transmembrane helix and lipid
hydrocarbon chain orientation. The schematic model shown
in Fig. 10 represents an attempt to visualize the
structural consequences of a-opioid receptor interaction
with either agonist or antagonist based on these
observations. Such a multi-state model allows a simple
explanation of the well-known fact that competitive
antagonists, although they occupy the same binding site in
the receptor as agonists, do not transduce signals across
the proteolipid membrane.
A more complete understanding of the molecular mechanisms
of receptor-ligand interactions will reqyre more detailed
information about the structural changes induced in the
receptor by different classes of ligands. In particular,
further time-resolved studies are needed to characterize
the sequence of conformational changes associated with the
intermediate states that follow ligand binding: It will
also be important to increase knowledge of the effects of
lipid membrane structure, salt concentration, pH, other
ligands such as allosteric effectors, and other proteins
(e.g. G-proteins, kinases, etc.) on the f4rmation of the
Iiganded states of the receptor.
The present method has shown that CPWR spectroscopy
provides a new and powerful experimental tool for such
investigations, for GPCRs, as well as other membrane-bound
receptors, enzymes, ion channels. In addition, the method
herein described should be readily adaptable to high
throughput screening, in view of the minute amounts of
receptor and ligand needed for a complete dose-response
CA 02413081 2002-12-16
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binding assay and for evaluation of receptor structural
changes.
Table 1: Dissociation constant values obtained'for DPDPE
5 and NTI obtained from experiments described in Figures 8
and 9.
DPDPE _ NTI
KD W ~ KD W'
1Q
DPDPE added first 10.0 ~ 0.4* 0.10 ~ 0.01**
NTI added first 40.0 ~ 0.4*** 0.020 ~ 0.005****
15 *No NTI present.
**Obtained in the presence of 80 nM DPDPE.
***Obtained in the presence of 0.14 nM NTI.
****No DPDPE present.
As would be understood by those skilled in the art, any
number of functional equivalents may exist in lieu of the
preferred embodiment described above. Thus, as will be
apparent to those skilled in the art, changes in the
details and materials that have been described may be
within the principles and scope of the invention
illustrated herein and defined in the appended claims.
Accordingly, while the present inventive method has been
shown and described in what is believed to be the most
practical and preferred embodiment, it is recognized that
departures can be made therefrom within the scope of the
invention, which is therefore not to be limited to the
details disclosed herein but is to be accorded~the full
scope of the claims so as to embrace any and all
equivalent products.
