Molecular Mechanisms of Peptide Loading by the Tumor Rejection
Antigen/Heat Shock Chaperone gp96 (GRP94)*
Srin
Sastry
and
Nora
Linderoth
From the Laboratory of Molecular Genetics, The Rockefeller
University, New York, New York 10021
 |
ABSTRACT |
Complexes of gp96/GRP94 and peptides have been
shown to elicit immunogenicity. We used fluorescence to understand
peptide association with gp96. A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low
salt (20 mM NaCl), the peptide binds gp96 with a
strong affinity (~100-150 nM) and forms pyrene excimers,
suggesting that the peptides were assembled as dimers. In high salt
(2.2 M NaCl), although peptide binding was stronger
(Ka
55 nM) than in low salt, pyrene
excimers were absent, implying that peptides were farther apart in the
complex. Heat shock-activated peptide binding exhibited characteristics
of both low salt and high salt modes of binding. Anisotropy and average
lifetime of the bound pyrene suggested that peptides were probably
located in a solvent-accessible hydrophobic binding pocket in low salt,
whereas in high salt, the peptide may be buried in a less hydrophobic
(more hydrophilic) environment. These results suggested that
peptide-gp96 complexes were assembled in several different ways,
depending on the assembly conditions. Resonance energy transfer between
the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or
more tryptophan residues were within the critical Forster distance of
27-30 Å from the pyrene in the bound peptide. It is proposed that
peptides are assembled within higher order gp96 complexes (dimers,
etc.) in a hydrophobic pocket and that there may be a conformational
change in gp96 leading to an open configuration for peptide loading.
 |
INTRODUCTION |
Heat shock proteins purified from tumor cells when injected into
inbred mice bearing the same specific type of tumor elicit immune
response against only that specific tumor (1-4). This paradigm, which
has been extended to virus-infected cells (5-7), as well as to a
variety of animal cancers (4), forms the basis for a new therapeutic
strategy against human cancers using
HSPs1 as vaccines. The
HSP-mediated specific immunogenicity has been attributed to endogenous
peptides that are noncovalently bound to the HSPs (8). Examples of
immunogenic HSP-peptide complexes include gp96 (GRP94), BiP, protein
disulfide isomerase, and calreticulin. These stress proteins are
abundant and are normally present in the lumen of the endoplasmic
reticulum (9). Interestingly, HSPs display dual functionality. On the
one hand, they act as molecular chaperones assisting in protein
folding, and on the other, they appear to shunt peptides into the
immune-response pathways. However, the relationship between the two
functions remains unclear. From our perspective, it is the immunogenic
function of HSP-peptide complexes that is the most intriguing and is of potential value to human health. The gp96 chaperone system is the most
extensively studied from an immunological standpoint (10-16). Gp96 is
an abundant endoplasmic reticulum resident glycoprotein that binds a
variety of peptides in vitro with no apparent amino acid
sequence specificity (17). Unambiguous confirmation of the immunogenic
properties of gp96-peptide complexes came with the demonstration that
in vitro reconstituted complexes were effective in
suppression of preestablished tumors (13). The HSP-mediated mechanisms
of immune response, although not yet fully understood, is an area of
very active research. One plausible pathway for peptide antigen
representation is outlined as follows. The antigenic peptides are
endogenously generated in the cytosol by proteases in the proteasome
either from precursor misfolded proteins or during extreme stress
caused by infection or the onset of cancer. These peptides are
translocated into the endoplasmic reticulum by the transporter
associated with antigen processing (18-21) and relayed on to HSPs,
which are known to be up-regulated during stress (22). The HSPs then
complex with the antigenic peptides and are recognized through a
receptor-mediated mechanism of macrophages or other specialized
antigen-presenting cells. The HSP-associated peptides are routed
through the endogenous presentation pathway in the antigen-presenting
cell and displayed in the context of MHC class I molecules. The peptide
antigens are finally recognized by the precursor CD8+
cytotoxic T lymphocytes (2, 23, 24). Exogenous antigen peptide
molecules chaperoned by gp96 can be presented by MHC class II molecules
and rerouted into the endogenous pathway of the MHC class I molecules,
a phenomenon referred to as cross-priming (3). These mechanisms provide
the underpinnings for the development of immunological strategies
against cancer and infectious diseases using HSP-mediated antigens (15,
16).
What is the mechanism of peptide loading by gp96? Only a few papers
have directly addressed this question in some molecular detail (13, 26,
27). It is important to understand peptide-gp96 interactions in order
to develop gp96 as an adjuvant for cancer immunotherapy. Here, we have
directly investigated the mechanisms of gp96-peptide interactions using
a fluorescence-based assay. The distinctive advantage of a fluorimetric
assay is that it is highly sensitive and yields quantitative
information. We report that gp96 binds a modified peptide with
surprisingly good affinities, the magnitude of which depends on the
binding conditions. We propose that two peptides may be bound extremely
close to each other in a gp96 dimer (or other higher order structures)
and that the peptides may be indeed bound in a heterogeneous microenvironment.
 |
EXPERIMENTAL PROCEDURES |
Chemicals and Modification of Peptide--
The pH of all buffers
was adjusted at 25 °C. Low salt binding buffer consists of 20 mM Na+-HEPES, pH 7.9, 20 mM NaCl, 2 mM MgCl2, whereas high salt buffer has 2.2 M NaCl, with other components being the same. 1-Pyrene butanoic acid, succinimidyl ester (catalog no. P-130) from Molecular Probes, Inc. (Eugene, OR), was used as recommended by the manufacturer. Peptide was purchased from Genosys Biotechnologies, Inc. (The Woodlands, TX) and purified by reversed phase C18 HPLC using a water/CH3CN/trifluoroacetic acid gradient. Purified peptide
was derivatized with 1-pyrene butanoic acid, succinimidyl ester as follows. To 0.39 mg of peptide dissolved in 0.1 ml of 100 mM NaHCO3 (pH 8.3) was added 1.0 ml of 5 mg/ml
1-pyrene butanoic acid, succinimidyl ester dissolved in
Me2SO. The mixture was vortexed and kept at room
temperature in the dark for 2 h. The reaction was quenched with
0.2 M Tris-HCl (pH 8.0) for 1 h and loaded on a
Sep-Pak C18 reverse phase cartridge (Waters). The peptide-pyr conjugate
was eluted with 100% methanol after washes, first with water and then with 60% methanol. The 100% methanol fraction was dried in
vacuuo and dissolved in 200 µl of 0.1 M TEAA (pH
7.0), and the peptide-pyr conjugate was purified by C18 reversed-phase
HPLC. The following HPLC gradient was used: 10 min of 0.1 M
TEAA-water and a linear ramp from 100% 0.1 M TEAA-water to
100% 0.1 M TEAA-60% CH3CN in 30 min. The
peptide-pyr-containing fractions were identified using in-line
absorption spectra collected using a diode array detector (1090 series
HPLC) and fluorescence spectra. The 1:1 ratio of pyrene to peptide in
the conjugate was confirmed by matrix-assisted laser desorption
ionization time of flight mass spectrometry (Protein/DNA Technology
Center, The Rockefeller University) and by the fluorescence spectra.
The concentration of peptide-pyr conjugate was determined using the
extinction coefficient of pyrene
340 4.7 × 104 M
1 cm
1. BA-pyr
was prepared as described above except that a 10-fold molar excess of
BA was added to 1-pyrene butanoic acid, succinimidyl ester.
Purification of His-tagged gp96--
A plasmid vector (pZ010)
was constructed by Dr. Z. Li in the laboratory of Dr. P.K. Srivastava
(University of Connecticut at Farmington, CT) and donated to us. This
plasmid contained the murine gp96 gene with a hexahistidine
tag inserted at its N terminus. In this construct, the
His-gp96 gene was transcribed in vivo by T7 RNA
polymerase, the synthesis of which was induced by
isopropyl-
-D-thiogalactopyranoside. Escherichia
coli JM109 (pZ010) was grown to an A660 of
0.8 at 37 °C in rich medium containing ampicillin (50 µg/ml).
Isopropyl-
-D-thiogalactopyranoside (0.5 mM)
was added to the growth medium, and the cells were infected with
bacteriophage M13-T7 gene1 (Invitrogen, Carlsbad, CA). Cells were harvested after 3 h, and all subsequent steps were performed at 4 °C. Cell paste was resuspended in 20 mM sodium
phosphate (pH 7.8), 50 mM NaCl, 1 mM
benzamidine, and lysozyme was added (100 µg/ml). The mixture was
stirred for 1 h and then subjected to three freeze-thaw cycles.
Following sonication, the lysate was adjusted to 10 mM
MgCl2 and 5 µg of deoxyribonuclease I per ml
(Worthington, Lakewood, NJ). The mixture was incubated for 1 h,
and the unbroken cells and debris were removed by centrifugation (20,000 × g for 30 min). Supernatant proteins were
precipitated overnight with ammonium sulfate (60% saturation). The
precipitate was centrifuged (20,000 × g for 20 min),
and the pellet was resuspended in Buffer IA (20 mM sodium
phosphate (pH 7.8), 500 mM NaCl, 20 mM
imidazole, 1 mM benzamidine). The centrifugation was
repeated, and the supernatant was loaded onto metal chelating Sepharose beads (Amersham Pharmacia Biotech) that were charged with
Ni2+ (0.1 M NiSO4). The column was
washed with 10 volumes of Buffer IB (Buffer IA containing 0.2% w/v
CHAPS) and 15 volumes of Buffer IC (IB with 80 mM
imidazole, pH 6.0). Bound proteins were eluted in Buffer ID (IC with
500 mM imidazole), and 5 mM EDTA was added to
the protein fractions to chelate any leached Ni2+.
Fractions containing gp96 were identified by SDS-polyacrylamide gel
electrophoresis followed by silver staining. Peak fractions were pooled
and diluted with Buffer IIA (sodium phosphate (pH 7), 17 µg/ml
phenylmethylsulfonyl fluoride) until the conductivity was equal to that
of Buffer IIB (Buffer IIA with 150 mM NaCl) and loaded onto
Q-Sepharose (Amersham Pharmacia Biotech). The column was washed with 20 volumes of Buffer IIB and 4 volumes of Buffer IIC (with 300 mM NaCl), and gp96 was eluted with Buffer IID (with 500 mM NaCl). Fractions containing gp96 were identified by
SDS-polyacrylamide gel electrophoresis (10% gel) and silver staining,
and these were pooled and concentrated to 1 mg/ml protein in a 50,000 Da molecular mass cutoff device (Amicon Inc., Beverly, MA). In some
instances, peak fractions of column 1 were used after pooling and
dialysis in 75,000 molecular weight cutoff membranes versus
Buffer IE (20 mM sodium phosphate (pH 7.8), 300 mM NaCl); sometimes 0.05% CHAPS was included in the
dialysis buffer. Using these procedures, we routinely obtained >95%
pure His-gp96. Yields were about 6 and 0.5 mg of gp96 from 1 liter of
cells following 1 and 2 columns, respectively.
Analyses of Proteins--
Purified gp96 was run on 10%
SDS-polyacrylamide gels. Marker proteins from Bio-Rad or Amersham
Pharmacia Biotech were run alongside. Gels were stained with Silver
Stain Plus reagents (Bio-Rad). Proteins were blotted onto
polyvinylidene fluoride membranes (Schleicher and Schuell) in chilled
transfer buffer (25 mM Tris base, 192 mM
glycine, 20% methanol) for 1 h at 70 V in a Mini Trans-Blot cell
(Bio-Rad). Membranes were rinsed with TBST (10 mM Tris (pH 8), 150 mM NaCl, 0.05% w/v Tween-20), blocked in TM (TBST
plus 5% dried skim milk) for 40 min and incubated with primary
antibody in TM for 3 h to overnight and then rinsed four times in
TM. The membrane was incubated with secondary antibody in TM for
1.5 h and rinsed six times with TBST. The following antibodies
were used: anti-grp94 and anti-grp78 (Stressgen, Victoria, British Columbia, Canada), anti-XpressTM (Invitrogen), sheep
anti-mouse Ig horseradish peroxidase conjugate (Amersham Pharmacia
Biotech), and goat anti-rat Ig horseradish peroxidase conjugate (Roche
Molecular Biochemicals). Detection was by enhanced chemiluminescence (Pierce).
Fluorescence Spectroscopy--
All spectra/measurements were
acquired at 23 °C using a computer-controlled JOBIN YVON/SPEX
fluorolog3
instrument (Instruments, S.A. Inc., N.J.) equipped with
polarizers. When required, the instrument was switched between the
steady state mode, with or without the polarizers engaged, and the
lifetime mode. The instrument was calibrated using Xe lamp intensity
spectra and water Raman emission spectra every time before measurements
were taken. The reference and experimental samples were loaded in fused
quartz square jacketed cuvettes (0.4 cm, 0.7 ml) with constant
stirring. The excitation and the emission bandwidths were set at 5 nm.
Excitation was usually at 340 nm, and emission was either at 376 nm or
in the scan mode, as specified in the figure legends. All steady state
emission spectra were corrected for wavelength-dependent Xe
lamp output intensity fluctuation and for the photomultiplier response
using a reference detector. Short integration times and an average of
three scans were used to minimize photobleaching of the fluorophore.
Standard emission acquisition was carried out in a final volume of 600 µl containing a constant concentration of peptide-pyr, His-gp96, or
BA-pyr (see figure legends). In the titration experiments, the maximum
volume change was 5-10% of the total sample volume. The data for the
binding isotherms is reported in an arbitrary scale, where the units
represent the corrected fluorescence Fpep
FBA, which indicates a value obtained after subtracting the
fluorescence intensity of BA-pyrene added to gp96 (control sample) from
the fluorescence intensity of the peptide-pyr + gp96 (experimental
sample). The calculation of the macroscopic binding constant was based
on a simple minimalist model of the reaction, as follows,
|
|
|
|
where G is His-gp96, P is peptide-pyr,
GP is the complex, and k1 and
k
1 represent the forward and the reverse rate constants, respectively. The macroscopic association constant (Ka) is given by the following relationship.
|
(Eq. 1)
|
[GP] is assumed to be directly proportional to the
corrected fluorescence intensity. A plot of the initial data points of the corrected fluorescence intensity versus [G]
or [P], with one or the other kept constant, would yield a
straight line with a slope that is equivalent to
Ka.
Anisotropy measurements were carried out using the T-format two-channel
method. The emission was measured with the S-detector set at 376 nm,
and the T-detector was fitted with a neutral density filter to cut off
stray light below 360 nm (Mellisgriot 03FNQ022). The alignment of the
polarizers was checked using a dilute solution of glycogen in water as
a scattering agent (anisotropy = 0.99807). The measurements were
done in the Instruments SA constant wavelength analysis mode by
excitation at 340 nm. The G-factor was measured, and the data were auto
corrected for dark current. Anisotropy (r) was calculated by
the computer program using the relation (28),
|
(Eq. 2)
|
where I is the fluorescence intensity with the
polarizers oriented either vertically (V) or horizontally
(H), and G is the instrument factor. Each
anisotropy measurement reported was the average of five independent
readings (S.E. = 0.005).
Average lifetime measurements were carried out using the phase shift
and demodulation method (28). The instrument was calibrated using
p-Terphenyl (average lifetime, 1.0 ns) and
(1,4-bis[5-phenyl-2-oxazolyl]benzene) (average lifetime, 1.3 ns) and
a scattering agent, LUDOX® TMA colloidal silica (lifetime, 0 ns).
Excitation was set at 340 nm, and emission was measured using the
T-detector fitted with the same filter used for measuring anisotropies
and the internal R-detector. The AC/DC signals from the reference
compound (glycogen) and sample were adjusted to approximately the same
ratios before the actual measurements were taken. Twenty-two
frequencies were used between 1 and 10 MHz in the log mode, with a
minimum of 5 and a maximum of 10 measurements. Phase angle varied
between 0 and 90°, and the modulation was between 0 and 1.0. The
residuals for the phase angles were within ±1.5, and for the
demodulation, they were within ±0.02. The data were analyzed using an
Instruments SA graph, and the average lifetime was calculated using
both the phase shift and demodulation data. Lifetime modeling was
carried out using a built-in postexperiment modeling application,
Lifetime, Version 2.1, assuming a multiexponential decay (see under
"Results"). Fitting the data to a single-component model or a
two-component model gave unacceptably high
2 values. We
used the three-component model because there are three major substances
in the reaction, viz., His-gp96, peptide, and pyrene. Using
the three-component model, better
2 values were
obtained. To achieve the lowest
2 values, Occam's razor
was applied for changing the concentration of each component in the
three-component model to obtain the best fits.
Dynamic Light Scattering--
was done using a
DynaProTM light scattering photon counting instrument
developed by ProteinSolutionsTM with an Nd-YAG laser as the
light source (883 nm). A built-in computer software package
automatically calculated the mean hydrodynamic radius (Rh) and the
average molecular mass of the protein aggregates using mono-model
dispersion, and the data were presented as histograms. Each molecular
parameter was an average of 40 individual measurements.
 |
RESULTS |
Purification of His-gp96 and Modification of a VSV Peptide with
Pyrene--
The His-tagged gp96 protein was overexpressed in E. coli, remained in a soluble form, and was purified by a two-column
chromatography procedure (see under "Experimental Procedures"). The
first column consisted of a Ni2+ affinity resin, which
specifically adsorbed the His-tagged gp96 protein. To further purify
the protein, we used an anion exchange resin. The final preparation was
greater than 95% pure His-gp96, as judged from a silver-stained
SDS-polyacrylamide gel (Fig. 1, top
panel). The His-tagged gp96 protein migrated slightly more slowly
than the intact murine gp96 preparation, presumably due to the addition
of the hexahistidine tag. Specific antisera were used to ascertain the
identity and intactness of the purified His-gp96 protein (Fig. 1,
bottom panels). Both murine gp96 and His-tagged gp96 were
recognized by antisera raised against mouse gp96, indicating that the
bacterial gp96 had the same epitopes as murine gp96 (Fig. 1,
bottom center panel). The intactness of His-gp96 was
confirmed by staining with two distinct antisera. The anti-Xpress
antibody recognized the bacterial plasmid-encoded amino acids in the N
terminus of His-gp96, but not the murine gp96 (Fig. 1, bottom
left panel). On the other hand, the C terminus of both proteins
was recognized by the anti-KDEL antibody, which recognized the
endoplasmic reticulum-localizing signal (Fig. 1, bottom right
panel). Unless otherwise stated, all the studies presented here
were done with His-gp96.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Characterization of purified His-gp96
protein. Top panel, 10% acrylamide-SDS gel stained
with silver. Left lane, His-gp96 purified in this
work; center lane, mouse gp96; right lane,
protein markers (from Bio-Rad). Bottom panels represent
immunoblots of His-gp96 and mouse gp96 proteins probed with various
antisera. Left panel, anti-Xpress, specific for the N
terminus. Center panel, anti-gp96 antiserum. Right
panel, C terminus-recognizing antiserum (anti-KDEL). One hundred
ng of gp96 was used per lane except where noted.
|
|
An immunogenic peptide (SLSDLRGYVYQGLKSGNVS) derived from the
nucleocapsid of VSV (29) that is known to be chaperoned by gp96 (3, 13,
14, 26) was synthesized by automated solid phase methods and purified
by reversed phase HPLC. We covalently attached a pyrene moiety to the
unique lysine via the
-amino group (Fig.
2) using a commercially available pyrene
succinimidyl ester derivative (30) (see under "Experimental
Procedures"). The expected molecular mass (m/z = 2445) of the peptide-pyrene conjugate (hereafter referred to as
peptide-pyr) was confirmed by matrix-assisted laser desorption
time-of-flight mass spectrometry (observed mass m/z = 2446). Pyrene is an excellent fluorescent probe for studying molecular
interactions because of several intrinsic properties (e.g.
Refs. 31-35). Its spectral characteristics are sensitive to the
environment, and it has an extremely long singlet excited state
lifetime (~100 ns), which makes it especially useful for studying
macromolecular motions. Its high photostability and high molar
absorptivity (
342 = 4 × 104
M
1 cm -1) and fluorescence
quantum yield (
383 = 0.25; sensitivity =
max
F 100 × 10
2) can
deliver very high signal to noise ratios. Fig.
3A shows the typical
excitation and fluorescence emission characteristics of the peptide-pyr
conjugate. These spectra are identical to the published spectra for
pyrene (30). The principal excitation band we used is at ~340 nm. The
structured emission shows the characteristic two major bands of pyrene
monomer: a band at ~376 nm that was slightly more intense than the
band centered at ~394 nm. Where applicable, the 376 nm band was used
to measure the emission intensity.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Amino acid sequence of the peptide used in
this work. The conjugation of pyrene moiety to the unique lysine
residue in the peptide via the -amino group is
figuratively shown.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
A, normalized excitation and emission
spectra of peptide-pyr. For the excitation spectrum, the emission was
at 376 nm. For the emission spectrum, the excitation was set at 340 nm.
B, changes in pyrene emission spectra as a consequence of
the binding of peptide-pyr to His-gp96. Gp96 and peptide-pyr were mixed
in either low salt buffer or high salt buffer and incubated at room
temperature for 30 min. The mixture was then dialyzed against a
1000-fold excess volume of the respective buffer to remove any unbound
peptide using a molecular weight 70,000 cutoff membrane. The emission
spectra of the samples were taken by excitation at 340 nm, and the
spectra were plotted using the same scale for all the scans. The molar
ratio of His-gp96 to peptide-pyr was 4:1 before dialysis. The
concentrations of peptide-pyr for the low salt and the high salt
experiment were 1.5 and 2.5 µM, respectively. The 2-nm
red shift in high salt is not readily apparent because of the long
scale used. Digitization of the spectra revealed the red shift (not
shown).
|
|
Binding of Peptide-Pyrene to gp96 in Vitro at Room
Temperature--
To ascertain that His-gp96 binds peptide-pyr, it was
incubated with a molar excess of His-gp96 for 10 min at room
temperature in low salt buffer (20 mM HEPES, pH 7.9, 20 mM NaCl, 2 mM MgCl2). The mixture
was dialyzed using a 70,000 molecular weight cutoff membrane for 2 h to remove any unbound peptide-pyr. The fluorescence of the retentate
was monitored by excitation at 340 nm (Fig. 3B). At this
excitation wavelength, the protein chromophores (Trp and Tyr) do not
absorb UV light, and only the pyrene is excited. We observed a striking
~1.8-2-fold increase in the intensity of the emission bands at 376 and 394 nm compared with that of an equimolar concentration of the
unliganded peptide-pyr. This indicated that the increase in emission
intensity must be due to interaction, i.e. the loading of
peptide-pyr by His-gp96. It has been previously shown that peptide
binding to gp96 could also occur in high salt buffers (13). In 2.2 M NaCl buffer, when peptide-pyr bound to His-gp96, the
pyrene emission intensity decreased slightly and the emission maximum
of peptide-pyr was slightly red-shifted by 2 nm (Fig. 3B)
compared with the unliganded peptide-pyr in high salt buffer (Fig.
3B). These results were reproducible, confirming that
changes in pyrene emission intensity was a convenient way of monitoring
and quantitating the binding of peptide to His-gp96. In addition,
because the emission intensity changed in both directions depending on
the salt concentration, it is likely that peptide-pyr interacts with
His-gp96 in at least two qualitatively different ways, depending on the
ionic strength of the buffer. To confirm that fluorescence changes
required the interaction of peptide with gp96, we repeated the above
experiments in low and high salt buffer with a BA-pyrene conjugate
(i.e. pyrene without the peptide) alone or with gp96.
Neither an increase nor a decrease in emission intensity was observed
at 376 or 394 nm (data not shown), indicating that changes in pyrene
emission in the earlier experiments were due to the binding of peptide
to His-gp96. Peptide binding appeared to be specific in the following
sense. The presence of a 100-fold excess of unmodified peptide in a
mixture of peptide-pyrene plus His-gp96 prevented changes in the
emission intensity, suggesting that the unlabeled peptide competed with
peptide-pyr for binding to His-gp96 (not shown). Furthermore, the
binding of peptide-pyr was saturable (see below). There was decreased
fluorescence emission intensity and a 2-nm red shift when peptide-pyr
was complexed with murine gp96 in high salt buffer, similar to that
seen with His-gp96 (Fig. 4). These
experiments demonstrate that fluorescence emission of pyrene is a
sensitive probe for studying peptide binding to gp96 and that the
bacterial His-gp96 behaved in an identical manner to the murine protein
under the same assay conditions.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
A, binding of mouse gp96 to peptide-pyr
in high salt buffer. A fixed concentration of peptide-pyr (140 nM) was titrated with increasing amounts of mouse gp96.
Final concentrations of gp96 were 0, 0.1, 0.2, 0.3, 0.4, 1, 3, and 10 µM. The curves obtained with the highest and lowest
values are noted. The excitation was set at 340 nm. The peptide-pyr
sample was incubated at room temperature in the fluorescence cuvette
for 10 min. Samples were then prepared by adding different amounts of
gp96 to the peptide-pyr solution and further incubated at room
temperature for 5-10 min before the emission spectra were taken. All
spectra were plotted to the same scale. The 2-nm red shift in high salt
is not readily apparent due to the long scale used. Digitization of the
spectra revealed the red shift. B, a plot of the initial
data points fitted to a linear regression line. The corrected
fluorescence intensity (arbitrary scale) was obtained by subtracting
the BA-pyr (control) intensity from the peptide-pyr intensity.
|
|
Different Modes of Peptide Binding to His-gp96--
A constant
amount of His-gp96 was titrated with various amounts of peptide-pyr
(Fig. 5A) under equilibrium binding
conditions. There was an increase in fluorescence yield relative to
parallel control experiments in which BA-pyrene was added to His-gp96
(see under "Experimental Procedures"). This increase reflected
binding of peptide-pyr to gp96, in agreement with the previous data
(Fig. 3). The corrected data were fitted to a linear regression
equation (Fig. 5A). Assuming pseudo first order kinetics
(see under "Experimental Procedures"), the slope of the straight
line was equivalent to the macroscopic association constant
(Ka
100 nm). In a reciprocal experiment, a
constant amount of peptide-pyr or BA-pyrene was titrated with varying
amounts of His-gp96 over a wider range of gp96 concentration (Fig.
5B). The binding behavior was biphasic. At lower
peptide-pyr/gp96 molar ratios (<0.5 µM His-gp96) the fluorescence intensity increased quite sharply (Ka
150 nM), similar to the previous result (Fig.
5A). However, at higher His-gp96 concentrations, the rate of
increase was slower (Ka
1 µM).
These experiments demonstrate that the assembly of peptide-pyr-gp96
complexes depends on the concentration of His-gp96 and that
interactions between gp96 molecules may result in higher order
assemblies, which could affect peptide binding.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Binding of His-gp96 to peptide-pyr in low
salt buffer. Excitation was at 340 nm, and emission was at 376 nm.
The corrected fluorescence intensity (arbitrary scale) was obtained by
subtracting the BA-pyr (control) intensity from the peptide-pyr
intensity. A, a fixed concentration of His-gp96 (1 µM) was titrated with increasing amounts of peptide-pyr
in low salt buffer. Titrations were performed in the fluorescence
cuvette as described for Fig. 4. B, titration of a fixed
concentration of peptide-pyr (0.15 µM) with increasing
concentrations of gp96 in low salt buffer.
|
|
Evidence for Higher Order Complexes--
To further investigate
the nature of the higher order His-gp96-peptide-pyr complexes, we
collected fluorescence emission spectra at high peptide-pyr/gp96 molar
ratios. The complexes were excited at 295 nm to monitor intrinsic
tryptophan emission from His-gp96. At this wavelength, the molar
absorptivity of tryptophan (
295
2 × 103 M
1 cm
1) is
greater than that of pyrene (
295
3 × 102 M
1 cm
1). In
addition, there are nine Trp residues in gp96. Because the emission
spectra of pyrene and tryptophan do not overlap, separate emission from
each compound should be observed. Fig.
6A shows an emission band
centered at 349 nm that corresponds to tryptophan emission. Tryptophan
emission in proteins ranges from
max at 308-352 nm,
depending on the local environment of the Trp residues, solvent
effects, excitation wavelength, and the extent of protein unfolding
(e.g. see Ref. 28 for a discussion). In addition, there were
emission peaks at 452 and 494 nm. These spectra were consistently
reproducible. Emission peaks at 452 and 494 nm are characteristic of
pyrene excimers (excited state dimers) (35-37). Surprisingly, no
pyrene monomer emission was detected (the Emax values of monomer are at 376 and 394 nm; e.g. see Fig.
3B). The pyrene excimer emission was detectable as a
function of increasing concentration of peptide-pyrene plus His-gp96
but not with peptide-pyr alone or with equal concentrations of
BA-pyrene conjugate plus His-gp96 (Fig. 6B). This result
indicates that peptide binding to gp96 was necessary for excimer
formation. Normally in solutions, pyrene excimers can be seen if
concentrations exceed 10
4-10
3
M pyrene (36, 37). However, in our system, the excimer was observed in 10
6 M concentrations and was
gp96-dependent (Fig. 6B). Because excimer emission requires that two pyrene moieties be hydrophobically stacked
face-to-face to form excited state dimers (e.g. see Refs. 36
and 38), our result suggested that peptides bound to gp96 were held in
close proximity to each other, probably in higher order
His-gp96-peptide-pyr complexes. Excitation of the complexes with the
340 nm absorption band of pyrene (
340
4.7 × 104 M
1 cm
1) also
showed the same two excimer species (compare Figs. 6A and 7A). Again, pyrene monomer
emission was quenched, suggesting that all peptides were stably bound
at these peptide/gp96 molar ratios.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
A, excimer formation in peptide-pyr-gp96
complexes. Emission spectra of intrinsic Trp residues and the excimers
in peptide-pyr + His-gp96 complexes in low salt buffer are shown.
Excitation was at 295 nm. Curve 1 was obtained with gp96
alone, and curve 2 was obtained with the highest
concentration of peptide-pyr (2.5 µM). The intermediate
curves were obtained with 0.04, 0.06, 0.09, 1.3, 1.6, 1.9, and 2.2 µM peptide-pyr. The concentration of gp96 was 10 µM. B, quantitation of the fluorescence
emission from Fig. 6A for pyrene excimers and Trp at their
respective emission maxima. Filled circles represent excimer
emission at 452 nm, and filled triangles represent excimer
emission at 494 and Trp emission at 349 nm. Open triangles
represent fluorescence intensity with mixtures of His-gp96 and
BA-pyrene (controls).
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Excimer emission spectra in the absence of
monomer emission. A, emission spectra of the excimers
in peptide-pyr + His-gp96 complexes were taken in low salt buffer.
Excitation was at 340 nm. Curve 1 is at the lowest
peptide-pyr concentration (0.03 µM), and curve
2 is at the highest concentration of peptide-pyr (2.5 µM). The intermediate curves were at 0.04, 0.06, 0.09, 1.3, 1.6, 1.9, and 2.2 µM. The concentration of gp96 was
10 µM. B, quantitation of the fluorescence
emission obtained from A for excimers at their respective
emission maxima.
|
|
Pyrene excimers are detected only when an excited state pyrene stacks
on to a ground state pyrene in a precise configuration during a
diffusive encounter. Generally, in solution at the time of light
absorption, the pyrene molecules are sufficiently dissociated such that
the excited state is localized only on one molecule (35, 36, 38). It is
interesting that such a precise excimeric geometry and flexibility was
achieved in the pyrenes when the peptides were bound to gp96. The
quenching of monomer fluorescence in these complexes suggests that
pyrene-pyrene interactions were not in equilibrium between stacked and
unstacked configurations. Normally, in aqueous solutions of pyrene
compounds, monomer emission and excimer emission are seen.
The absence of monomer emission has been observed in pyrene crystals
and in model pyrene compounds, such as glasses and pyrenophanes (two
pyrenes linked face-to-face by methylene groups), in which ground state
pyrenes are so close that they may be stacked (35). The photophysical
behavior of these model compounds suggested that in our case, two
peptide-pyr units were extremely close to each other in the
gp96-peptide complexes (~3.5-10 Å). Another facet of the higher
order structure of the complexes is that there were two excimer
species, as inferred from the presence of two excimer peaks (Figs. 6
and 7). To further probe the status of pyrene in the His-gp96 complexes
and to confirm the existence of the emissive species, we recorded the
excitation spectra of the complexes at the highest gp96 concentration
used (i.e. as in curve 2 of Figs. 6A
and 7A). Fig. 8A
shows that the excitation spectra at the excimer emission (at 451 or
494 nm; Fig. 8A) were different compared with the excitation
spectra taken at the monomer emission wavelength (Fig. 8B).
Such a difference is generally taken as compelling evidence for ground
state preassociation of pyrenes (35). For example, the longer
wavelength excitation bands of the monomer were suppressed, and a
single very broad excitation band centered at a
max of
228 nm (Fig. 8A). The excitation spectra are qualitatively
the same for both emission wavelengths, except that the excitation
intensity for the 452 nm emissive species is slightly greater than that
of the 494 nm emissive species (Fig. 8A). This is consistent
with results shown in Figs. 6A and 7A (emission
at 452 nm is greater than at 494 nm), and suggests that the population
of the shorter wavelength excimer is larger than that of the longer
wavelength excimer. When excitation was monitored either at 349 or at
376 nm, the spectra were different from the excimer excitation spectra
(compare Fig. 8A with Fig. 8B). (Excitation was
monitored at 376 nm, even though emission spectra showed no pyrene
monomer, partly to confirm its absence.) For example, the structured
bands that are normally seen between 320-343 nm for pyrene monomer
were coalesced into a single broad blue-shifted peak at 327 nm (Fig.
8B). The other excitation band, normally centered at 240 nm
in the monomer (Fig. 3A), was now broader and blue shifted
to 227 nm. However, the excitation band at 268 nm appeared rather
unchanged in its position but was considerably broader. Note that the
hyperchromic band at 227 nm was present in both excitation spectra
(Fig. 8), indicating that the same set of species was present. The
UV-visible absorption spectra confirmed the presence of intact pyrene
with the expected peaks at 265, 276, 325, and at 343 nm (not shown).
However, the absorption band at 343 nm appeared broader
(peak/valley = 1.87) compared with unassociated peptide-pyrene
(peak/valley = 2.57). Inner filter effects were nonexistent
because the A340 values of the samples were
<0.08. Collectively, these results suggest that complexes of
peptide-pyr and His-gp96 were heterogeneous. Although it is difficult
to definitively identify the local environment of the pyrene in this
complex system, it is possible that the emission band centered 494 nm
may result from a dynamic excimer, whereas the isoemissive peak at 452 nm may suggest a less dynamic, perhaps more static excimer. Based on
our knowledge of model pyrene compounds (35), we suggest that the
excimer with
max 452 nm is from pyrene dimers in a more
nonpolar microenvironment and that the excimer with
max
494 nm may represent dimers in a more polar microenvironment. We hasten
to add that stronger evidence may be needed to fully support this
suggestion. The excimer with
max 452 nm and that with
max 494 nm may have somewhat different pyrene
configurations, although the two may appear to have the same steady
state excitation spectra. The presence of only one major hyperchromic
excitation band (Fig. 8A) may suggest a very close ground
state preassociation of pyrenes. The excimers may originate from a
variety of configurations with nonidentical interchromophoric
distances. In the shorter wavelength excimer, the interchromophoric
distance may be shorter than in the longer wavelength excimer. In
support of our interpretations, we refer to previously recognized cases
of pyrene excimer heterogeneity in pyrene-labeled polyalanine (39) and
other model pyrene compounds (35). In the aforementioned studies, the
presence of two emissive excimer species, one of a shorter wavelength
than other, corresponded to pyrene in differentially polar
microenvironments. It is possible that there may be more than one
peptide-binding site in different microenvironments or the same binding
site but gp96 may adopt different conformations (see under
"Discussion"). To rule out the possibility that the excimers were
due to nonspecific effects, we conducted several control experiments.
Substituting BA-pyr for peptide-pyr did not result in excimer formation
(Fig. 6B, open triangles). We also investigated the effects
of binding of peptide-pyr to two other peptide-binding proteins,
viz., mouse albumin, a protein that is known to strongly
bind peptides, and glucose-6-phosphatase, a protein that very weakly
interacts with peptides. Using these two proteins and peptide-pyr, we
observed no excimer emission (Fig. 9).
Thus, peptide-pyr excimer formation results from a unique interaction
of gp96. In summary, our experiments indicated that gp96 appears to
load peptides at close proximity (~3.5-10 Å) in specific
orientations forming dimers.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
Excitation spectra of the excimers. The
peptide-pyr (2.5 µM) + His-gp96 (10 µM)
complexes in low salt buffer were scanned at the indicated emission
wavelengths (Em).
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 9.
Emission spectra of peptide-pyr (2 µM) complexed with either mouse albumin
(A) or with glucose-6-phosphatase (B)
in low salt buffer. The excitation was at 340 nm. Titration curves
were obtained with different concentrations of proteins (0, 2, 3, 4, 5, 6, 7, and 9 µM).
|
|
Resonance Energy Transfer between Intrinsic Tryptophan(s) and
Pyrene--
Upon excitation at 295 nm, the fluorescence emission
(
max 349 nm) of the intrinsic tryptophan(s) was
quenched, concomitant with an increase in excimeric emission intensity
(
max 452 and 494 nm; Fig. 6). A 2-fold quenching was
observed at high peptide concentration. Note that the broad emission
band for Trp (330-355 nm;
F = 0.2; sensitivity =
max
F 11 × 10
2)
overlaps with one of the strong excitation bands of pyrene centered at
340 nm (
340
4.2 × 104
M
1 cm
1). For this reason, the
relative excimeric emission intensities (Ex 295 nm) are
much higher than that of Trp (Fig. 6B). This, coupled with
the decreasing fluorescence intensity of Trp, indicated that Trp
fluorescence was quenched by pyrene. The quenching of the Trp emission
by pyrene is also inferred from the excitation spectra of the
pep-pyr-gp96 complexes (Fig. 8B). These observations are
consistent with a nonradiative energy transfer between the intrinsic
Trp residue(s) (donor) and pyrene (acceptor). Such resonance energy
transfer has been observed previously in systems in which proteins with
intrinsic Trp(s) were labeled with pyrene (see below for references).
Our data do not show which of the nine Trp residues in gp96 were
involved. However, for energy transfer to occur, one or more of the
Trps should be within the critical Forster distance of 27-30 Å from
the pyrene attached to peptide (28, 40-43).
Peptide Loading by Heat-induced Activation and in High Salt
Buffer--
It was previously demonstrated that peptide loading
occurred efficiently in high salt buffer (Fig. 4) or by heating the
peptide-gp96 complexes to 50-60 °C and then cooling to room
temperature (13, 26). We wished to test whether peptide binding to gp96
differed qualitatively under these conditions as compared with the low salt equilibrium conditions (Figs. 5-7). Fig.
10 shows that unlike the case with low
salt conditions (20 mM NaCl; Figs. 3 and 5), the
fluorescence emission intensity was quenched with increasing gp96
concentration in 2.2 M NaCl. In addition, there was a small but reproducible 2-nm red shift in the emission maxima of the two
peaks, suggesting that the pyrene may be located in a more hydrophilic
environment. This result is consistent with the earlier observation
with mouse gp96 (Fig. 4), indicating that His-gp96 and mouse gp96 load
peptides in a similar manner. Surprisingly, all emission was from
pyrene monomer (Figs. 4 and 10A). No excimer occurred in
high salt even at very high gp96 to peptide ratios, although the
monomer emission was quenched. This indicated that the bound peptides
in the complexes were arranged in a different manner in high salt
conditions as compared with low salt. Based on the initial binding
isotherm and using curve fitting by linear regression, we estimated
that the macroscopic binding constant under high salt conditions was
equivalent to
55 nM (Fig. 10B). This
estimate suggested that the gp96 binds peptide-pyr about 2-3-fold more
efficiently in high salt than in low salt, consistent with an earlier
observation (13). From the initial binding data points for mouse gp96,
we estimated that the mouse gp96 protein appeared to bind peptide-pyr
with an estimated macroscopic binding constant equivalent to ~700
nM (Fig. 4B). This suggested that in our assay,
the His-tagged bacterial protein bound significantly better than the
mouse protein in high salt buffer (see under "Discussion").

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 10.
Binding of His-gp96 to peptide-pyr in high
salt buffer. A, emission scans were taken with
excitation at 340 nm. The concentration of gp96 was varied from 0 (top curve) to 3.5 µM (bottom
curve), whereas the concentration of peptide-pyr was held constant
(2 µM). The intermediate curves were obtained with 3.5, 7, 10.5, 13.5, 20.5, 27.5, 34.5, 41.5, 48.5, 55.5, 66.0, 78.5, 87, 97.5, 147.5, 197.5, 297.5, 447.5, 647.5, 847.5, and 1047.5 nM and 2.2 µM. The 2-nm red shift in high
salt is not readily apparent because of the long scale used.
Digitization of the spectra revealed the red shift (not shown).
B, plot of the initial data points obtained from
A for the binding of His-gp96 to a fixed concentration of
peptide-pyr (150 nM).
|
|
Neutral salts influence protein conformational transitions (shapes) and
aggregated states because of their electrostatic and lyotropic effects.
Electrostatic effects may depend on the ionic strength and sign of the
charge. Lyotropic effects are mostly independent of the charge. These
effects may stabilize or destabilize the native structures of
macromolecules as well as alter the structure of water (44). In order
to glean whether there are any qualitative changes in the higher order
of His-gp96 molecules under the low salt and high salt conditions (20 mM and 2.2 M NaCl, respectively), we used
dynamic light scattering to estimate the relative aggregate sizes.
His-gp96 formed very large aggregates as measured by the mean molecular
mass and hydrodynamic radius (Rh), consistent with an earlier report
(45). Here we used light scattering only as a semiquantitative tool to
determine whether the aggregated state of gp96 varied in the different
salt concentrations. Dynamic light scattering gives only rough
estimates but not accurate measurements of the molecular mass and size
of the aggregates. Moreover, because gp96 is probably not a globular
protein and may be oddly shaped or elongated, as demonstrated by more
extensive previous studies (45), our measurements may have been
influenced by shapes of the oligomeric structures. In 20 mM
NaCl, the mean mass of the aggregates was 8717 ± 38 kDa with a
radius Rh = 28.98 nm. In 2.2 M NaCl, the mean mass of
the aggregates was 15441 ± 87 kDa with an Rh = 33.98 nm.
High salt caused a 77% increase in the aggregated mass and a 16%
increase in the extended state. These results indicated that the
His-gp96 protein appeared to be more polydispersed and extended in high
salt. The absence of excimer in high salt could be related to these
changes in gp96 conformation. The bound peptides may be farther apart
as compared with those in low salt condition. However, the absence of
excimer fluorescence does not necessarily mean that the pyrenes are not
stacked, nor does it indicate that peptides are not close to each
other. We can only conclude that in high salt the pyrenes are not
oriented in an excimeric geometry. Deviations from the precise geometry
of stacked pyrenes may quench the excimer fluorescence. One possibility
can be excluded based on the light scattering data. Excimers in low
salt are probably not due to random aggregation of gp96 molecules. If
this were the case, excimer would have been observed in high salt as
well, where aggregation was greater, as indicated by dynamic light
scattering. We cannot rule out the possibility that high NaCl
concentration itself may have quenched the excimer emission. However,
salt-dependent fluorescence quenching is usually observed
with exciplexes in nonpolar media with quaternary ammonium salts (46),
conditions completely unlike those used here.
Next, we examined the loading of peptide-pyr under heat shock
conditions. Peptide-pyr plus His-gp96 mixtures at a molar ratio of 1:1
or 2:1 were prepared and heated to 60 °C for 10 min and cooled to
room temperature. The mixture was then passed through a Sephadex G25
column. The complexes and any uncomplexed His-gp96 were recovered in
the void volume, and any free peptide was trapped in the column. The
presence of gp96 in the void was verified by Western blotting with
anti-Xpress antisera (although not shown, see, for example, Fig. 1).
The fluorescence emission spectra showed that whereas the excimer band
centered at 494 nm was present (Fig. 11, curves 2 and
3), it was less prominent than in simply low salt conditions
(Fig. 6A). The monomer emission decreased slightly at 378 nm
and quite dramatically at 396 nm. Note that there was a 2-nm red shift
of the emission maxima, as previously shown in the case of high salt
binding. This result indicated that heat-activated His-gp96 bound
peptide-pyr in a manner that is in certain ways reminiscent of both low
salt and high salt binding. An excimer was seen, as in the previous
case of low salt binding (Fig. 6A), and there was a decrease
in monomer emission as in the case of high salt binding (Fig. 10).
However, an important difference is that only the classic polar
(dynamic) excimer was observed (
max 494 nm). A
noteworthy fact is that the excimer was observed even at equimolar
ratio of peptide-pyr:His-gp96, suggesting either that some His-gp96
molecules may carry more than one peptide or that two gp96 molecules,
each with a peptide, may form dimers, whereas others carried no peptide
at all, assuming a second order reaction. Thus, there may be at least
two peptide-binding sites in gp96, or alternatively, each gp96, with
one peptide, may form a dimer. Previously published data (45) appear to
favor the latter scenario (see under "Discussion").

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 11.
A, heat shock-induced binding of
peptide-pyr to His-gp96 in low salt. Emission scans were taken after
excitation at 340 nm. Mixtures of His-gp96 + peptide-pyr were assembled
either at a 1:1 molar ratio (curve 2) or at a 1:2 molar
ratio (curve 3) and heated at 60 °C for 10 min and cooled
to room temperature. The mixture was then passed over a Sephadex G25
column (1 ml) to remove any unbound peptide-pyr. The complexes were
present in the void volume, as ascertained by Western blotting using
anti-Xpress antibody and by the characteristic pyrene absorption
spectrum. Curve 1 shows peptide-pyr heat-shocked and cooled
to room temperature. The slight red shift in the emission peaks of the
complexes relative to peptide-pyr is noted. B, changes in
the tertiary structure of His-gp96 in low salt buffer or in high salt
buffer or after a heat-cool cycle. The intrinsic Trp emission spectra
are shown under the specified conditions. The same concentration (2.5 µM) of His-gp96 was used to acquire all the spectra. The
excitation was at 295 nm.
|
|
To understand the structural transitions in His-gp96 under the binding
conditions used in this work, we monitored the intrinsic Trp
fluorescence, which often gives clues about the tertiary structure of
proteins (48). The normalized fluorescence intensity of His-gp96 in low
salt was lower than that of either heat-shocked gp96 or gp96 in high
salt (Fig. 11B). The emission spectrum is slightly red-shifted for the heat-shocked gp96 and for gp96 in high salt (2.2 M NaCl), compared with the low salt gp96. These results
indicated that for each of the binding conditions tested here, the
protein tertiary structure was somewhat different. Because heat shock increased the Trp fluorescence intensity and slightly red-shifted the
emission spectrum compared with the native protein in low salt, the
protein may have adopted a more unfolded or solvent-exposed conformation after heat shock. In support of this, we have also observed increased protease susceptibility of the heat-shocked gp96
compared with the native protein (not shown), which has also been
reported by others (27). The emission spectrum in high salt and that
seen after heat shock bear some similarity except that the intensity is
higher in the former case. (Note that the protein concentration is the
same in all spectra.) This similarity suggested that there could be
some conformational similarity after heat shock and in high salt. This
may explain our earlier observation that there was a decrease in pyrene
fluorescence intensity in both high salt-induced binding and in heat
shock-activated binding.
Anisotropy and Lifetime--
To gain greater insights into the
state of pyrene in peptide-His-gp96 complexes, we measured the steady
state fluorescence anisotropy of pyrene. Anisotropy is directly related
to the rotational correlation time through the Perrin equation (see
Ref. 28 for a discussion).
|
(Eq. 3)
|
where r0 is the limiting anisotropy (no
rotation), r is the steady state anisotropy,
is the
fluorescence lifetime; and
is the rotational correlation time of
the fluorophore.
Our experiments were done either in low or in high salt in the absence
or the presence of a large excess of unlabeled peptide (competition
experiment). Fig. 12A shows
that upon addition of gp96 to a fixed concentration of peptide-pyr, the
fluorescence anisotropy increased due to complexation in both low salt
and high salt. Going from the lowest to the highest concentration of
gp96, the change in anisotropy was larger in low salt buffer (filled squares) than in high salt buffer (filled
circles). This indicated that the time-averaged rotational
diffusive motions of the fluorophore, and hence the peptide, are more
restricted in low salt than in high salt. Addition of a molar excess
(10-100-fold) of unlabeled peptide to the bound complexes in low salt
buffer resulted in decreased fluorescence anisotropy, consistent with the release of the peptide-pyr through competition (Fig. 12A,
open circles). Scatchard analysis of the anisotropy data yielded a straight line, the slope of which is equivalent to the reciprocal of
the dissociation constant (Fig. 12B; Kd
260 nM). Comparing these results with Fig. 5, our data
suggest that the rate of peptide dissociation is roughly 2-3 times
slower than the rate of association in the low salt conditions.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 12.
Anisotropy of peptide-pyr + His-gp96
complexes. The anisotropy values (ordinate) were obtained by
subtracting the anisotropy values of a mixture of BA-pyr (0.15 µM) + different amounts of His-gp96 (control samples)
from the corresponding values obtained with a mixture of peptide-pyr
(0.15 µM) + His-gp96. The symbols indicate values
obtained with low salt buffer (squares), with high salt
buffer (filled circles), and with a molar excess (10-100×)
of unlabeled peptide in low salt (open circles).
B, Scatchard analysis of the anisotropy data from Fig.
11A. Ln bound anisotropy = Ln total anisotropy (free + bound) Ln anisotropy remaining after the addition of excess
unlabeled peptide (free).
|
|
Next, to further understand the interaction of the peptide with gp96 in
the context of the microenvironment of the binding site, we measured
the average lifetime of the bound pyrene in low salt and in high salt.
The average lifetime of pyrene was measured using the phase shift and
frequency modulation method, which yields an indirect measure of the
excited state lifetime (28). Table I
presents a summary of the average lifetimes of pyrene under various
experimental conditions. In our model, the relative concentration of
the third component (
3) was closest to unity and far
exceeded the concentrations of the other components, which were
negligible. For this reason,
1,
2, and
the corresponding
values were ignored, and the lifetime
(
3) of the third component was interpreted as that of
pyrene. This is consistent with the fact that in our system the only
excitable species at 340 nm was pyrene. Furthermore, the modeled
average lifetime of the third component (
3) does indeed
agree to a good approximation with the average lifetime literature
values for pyrene derivatives reported in various systems (80-100 ns
(30-32, 35)). Free pyrene (BA-pyr) had a shorter lifetime than
peptide-conjugated pyr. This may be because the fluorophore is more
restrained or less solvent accessible when conjugated to peptide. In
high salt, peptide-pyr had a much longer
than the free pyrene or
peptide-pyr in low salt, consistent with the known
environment-sensitivity of pyrene (30). The average lifetimes of the
peptide-pyr-gp96 complexes were measured at a fixed molar ratio of
peptide-pyr to gp96 (1:4). In low salt, whether the peptide was bound
to gp96 or not, the average lifetime of pyrene appeared to be more or
less the same within experimental error. However, in high salt the
lifetime changed significantly compared with the unbound peptide-pyr,
suggesting that the microenvironment of the peptide-binding pocket may
be more solvent-accessible. In high salt, the average lifetime of the
bound pyrene is shorter than that in low salt. Using the Perrin equation (Equation 3) we calculated the average rotational correlation time (
). In low salt,
was 68.64 ns, whereas in high salt,
was 24.78 ns, suggesting that the peptide may be less restrained in the
binding pocket in high salt and more susceptible to solvent-induced relaxation in high salt compared with in low salt. Because the average
lifetime is more or less the same in low salt whether the peptide is
bound or not, the fluorescence enhancement (Figs. 3 and 5) may be
attributable either to greater quantum yield and/or increased steady
state anisotropy. Although we have not measured the fluorescence
quantum yield, we suggest that the increased anisotropy (Fig.
12A) upon peptide binding to His-gp96 may have contributed
to the enhanced fluorescence. On the other hand, in high salt the
average lifetime of the bound fluorophore decreased consistent with
lower anisotropy values, implying that quantum yield and steady state
anisotropy may have contributed to the quenching of the fluorescence
(Fig. 10).
View this table:
[in this window]
[in a new window]
|
Table I
Average lifetime (in ns) of pyrene butylamine, peptide-pyr, and the
complexes with gp96
The standard deviation for the lifetime was ±2 ns. For each
experiment, 22 data points were used in the data fitting algorithm. NV,
no value: the algorithm produced either a value of 0 or negative
values.
|
|
 |
DISCUSSION |
In this paper, we have investigated the details of the mechanism
of binding of peptide to the tumor rejection antigen gp96 (GRP94) using
fluorescence spectroscopy. To our knowledge, there are only a few
published papers that have directly addressed the mechanism of peptide
loading by gp96 at the molecular level (13, 26, 27). Here, we have
utilized a pyrene-labeled VSV-derived peptide to study the structural
changes accompanying its binding to gp96. This peptide has been
previously shown to be a natural substrate for gp96 binding (13, 14).
Therefore, our results may have relevance to the specific immunogenic
properties of gp96 preparations from VSV-infected cells (3, 13) and, in
general, to the paradigm of heat shock proteins in the immunotherapy of cancer and infectious disease (4, 16, 47). Coupling the strongly
hydrophobic pyrene moiety to the peptide may have altered peptide
affinity for gp96. However, this potential caveat is more than offset
by the advantage of having an environment-sensitive probe (pyrene) for
monitoring peptide binding in a rapid and direct manner with very high sensitivity.
Our principal findings are as follows: 1) His-gp96 binds peptide-pyr
with reasonably high affinity (~0.1-1.0 µM) depending on the peptide/gp96 molar ratio. 2) There are several different modes
of peptide binding, and the microenvironment of the bound peptides may
be different depending on salt conditions or after heat shock
activation of gp96. 3) More importantly, in the bound state two bound
peptides are in close proximity. Previously reported binding constants
were significantly weaker (15 µM (26)) compared with our
results. This discrepancy may be due to a number of factors. The
peptide used in the previous study was a truncated version of our
peptide (only about half the size of our peptide) and had a cysteine;
and the presence of pyrene itself may increase peptide affinity by its
hydrophobic nature. The binding conditions were also different, and the
gp96 in the previous work was from mammalian cells, whereas our gp96
was a genetically engineered version from bacteria. The latter
difference may be important because mammalian gp96 may have peptides
that were preloaded and tightly bound, whereas our bacterial
preparation may have fewer and/or less tightly bound preloaded
peptides. This suggestion is supported by a comparison of the magnitude
of the fluorescence changes between mouse gp96 and His-gp96 and the
apparent macroscopic affinity constants of the two proteins under the
same assay conditions (Fig. 4 versus Fig. 10). The bacterial
protein appears to bind peptide-pyr significantly better than mouse
gp96 does. Peptide loading by His-gp96 is biphasic (Fig. 5). At lower
peptide/His-gp96 ratios the protein loads peptide with a greater
efficiency (Ka
100-150 nM) than at
higher gp96 to peptide molar ratios (Ka
1 µM), suggesting that gp96-gp96 interactions may compete
for peptide binding. This result agrees with dynamic light scattering
showing that gp96 forms higher order oligomers and with previous
results (45). At low His-gp96 concentration (2 µM), the
mean aggregate mass was 857 kDa (Rh = 22.47 nm), whereas at higher
concentration (8 µM) it was 8879 kDa (Rh = 28.85 nm), and at a still higher concentration (11 µM) it was
14,139 kDa (Rh = 33.15 nm). (These measurements were done in 150 mM NaCl, whereas the earlier measurements (see under
"Results") were done at a different salt concentration.) These
measurements only reflect the relative qualitative differences in the
aggregated state of gp96 and are not accurate dimensions of the
aggregates. It is unusual that gp96 aggregated even in micromolar
concentrations, unlike control proteins, bovine serum albumin, and
lysozyme, which displayed their approximate monomeric molecular masses
at each of the above mentioned concentrations. The binding affinity of
peptide-pyr to His-gp96 was 3-fold better in 2.2 M NaCl
(Fig. 10) than in low salt (Fig. 5A), consistent with a
previous report (13). However, our results reveal a new facet of gp96:
there is a fundamental difference in the way His-gp96 loads peptide in
low salt versus high salt. In low salt, peptides appear
bound in close proximity, probably as dimers, as revealed by pyrene
excimer formation. In high salt, the peptides may be bound farther
apart, because there was no excimer, although they may still be bound
as dimers. A physical separation of the peptides on gp96 may account
for the greater apparent macroscopic affinity. Our results can be
conveniently explained on the basis of published findings that gp96
exists as a dimer and in higher order oligomers. It has been proposed
that the gp96 molecule contained a C-terminal dimerization domain
(amino acid residues 692-709) and that the native protein is assembled
into rod-shaped tail to tail dimers (45). Electron microscopic studies
showed that the protein is highly extended (~11.5-28 nm) into large
dimers and rods or as "boomerang-shaped" (our term) oligomers. Our
dynamic light scattering measurements (see above) essentially confirm
these findings, although the size estimates differ somewhat from those
of Wearsch and Nicchitta (45), probably because we have used less
accurate techniques than those used by the previous authors. Assuming
that each gp96 molecule binds one peptide and the native protein exists
as a dimer, the excimeric configuration is possible through the close juxtaposition of two peptides with the pyrene dimers stacking on top of
each other. In the winged or boomerang configuration of the gp96 dimer,
two peptide-pyr molecules could be brought closer by the dynamic bend
in the dimer. The stably locked dimer configuration may facilitate the
static (nonpolar) excimer (
max 452 nm, Figs. 6 and 7),
whereas the bent boomerang gp96 dimer may produce the dynamic excimer
(
max 494 nm, Figs. 6 and 7). In the latter case, the
peptides may be brought closer by diffusive motions of the gp96 dimer.
At any rate, the existence of specific dimeric or oligomeric states of
gp96 is an attractive way to explain our results and our results
indirectly confirm the dimer hypothesis of the previous workers (45).
The propensity of gp96 to aggregate may also explain the decrease in
peptide binding at high gp96/peptide ratios. The peptide-binding
site(s) may be closed off or are less accessible in gp96 oligomers
because of site to site competition. It is tempting to speculate that
the C-terminal hydrophobic dimerization domains (45) may be close to
peptide-binding sites.
Heat-cool-induced loading of peptide probably occurred as a result of
protein unfolding. The unfolding during the heating step may release
prebound peptides so that extraneous peptides may be loaded.
Alternatively, heat-cool may induce a specific conformational change in
the tertiary structure, which facilitates peptide loading (Fig.
11B). Heat shock activation mimics aspects of both the high
salt and low salt type of binding modes (Fig. 11). However, an
important subtext is that the shorter wavelength (static) excimer was
absent. If the static excimer forms through the precise locking of gp96
dimers, it is not difficult to expect that such a precise excimeric
geometry might not be achievable after heat shock. Presumably, the
configuration of the dimer in the low salt complex is not exactly the
same as the heat-shocked complex. This notion is supported by the Trp
emission spectra (Fig. 11B), which showed that the two
conditions generated most likely nonidentical tertiary structures.
However, the heat shock treatment does not prohibit the dynamic
excimer, signifying a more flexible mode of peptide-carrying gp96
dimer. In our experiments, peptide binding through heat shock
activation appears to occur at much lower gp96/peptide molar ratios
than those previously reported with the mouse gp96 (13, 26). This may
be because bacterial His-gp96 is less saturated with endogenous
peptides than mouse gp96. The Trp emission spectrum (Ex at
295 nm) of His-gp96 appeared rather broader and slightly red-shifted
(
max 343 nm) compared with mouse gp96
(
max = 338 nm; not shown). This is suggestive of a more
unfolded His-gp96 than the mouse variety. (It is unlikely that these
differences are due to the hexa-His tag because the
max
of His is at 211 nm.) Interestingly, the UV-visible absorption of the
heat-cooled peptide-loaded His-gp96 was broader and showed a dramatic
hypsochromic shift of spectrum compared with that of the heat-cooled
unloaded protein. The peak/valley ratio of the loaded protein was 0.83, compared with 2.0 for the unloaded protein. These changes in the
absorption spectra of the liganded protein, coupled with the fact there
was quenching of the Trp emission intensity with the loading of the
peptide (Fig. 6), suggested that there may be a conformational change
in the peptide-bound gp96 molecule accompanying the loading of the
peptide. It is suggested that peptides bind to an open conformation of
gp96 in a hydrophobic pocket, and the loading of peptide leads to
conformational adjustment in the protein. This suggestion is in
agreement with that of Nicchitta and co-workers (27), based on
fluorescence changes using Nile Red. This peptide binding model is
analogous to other peptide binding systems such as MHC class II
(49-51). Purified native gp96 comes loaded with peptides, as indicated
by its immunogenic properties (25) and by its apparent mass as measured
by matrix-assisted laser desorption-time of flight mass spectrometry
(data not shown). In our hands, purified mouse gp96 showed two apparent
mass peaks, one at m/z = 98.8 kDa and another at 99.6 kDa (mass accuracy of 0.1%). This result indicated an additional mass
of ~3-4 kDa conferred by peptide. (The calculated mass from the
amino acid sequence of mouse gp96 is 92,475.98 Da. The extra mass (~ 96,000
92,475.98 = ~ 3525 Da) is from posttranslational
covalent modifications.) In contrast to other chaperone systems
(e.g. HSP70), in which adenine nucleotide regulates the
release of peptides, a native "empty" state of gp96 bereft of
peptides has not been documented. Indeed, it may be that with gp96 only
various transitions of "open" states may exist, wherein peptide
loading occurs by competition/mass action. (In this context, we have
not observed any significant changes in fluorescence intensity of
gp96-peptide-pyr complexes in the absence or presence of ATP or ADP
(not shown), in agreement with a previous report (26).) The environment
sensitivity of pyrene makes it a valuable tool for studying peptide
binding. The fluorescence yield of peptide-gp96 complexes increased in low salt and decreased in high salt. This modulation of pyrene fluorescence may suggest that the peptide binds in a more hydrophobic environment in low salt and in a more hydrophilic environment in high salt.
The observation of resonance energy transfer between the intrinsic Trp
and pyrene is interesting (Fig. 6) because it suggests that there may
be Trp residues in the peptide-binding pocket or, at the very least,
that one or more Trp residues are within the critical Forster distance
(27-30 Å) of pyrene. Of the nine Trp residues in gp96, six are
located on the N-terminal side (amino acids 5-343). Three of these are
clustered between residues 282 and 343. In addition, there are three
Trp residues between amino acid 485 and amino acid 554. However, in the
absence of a high resolution three-dimensional structure, it is
impossible to predict the relative position of these residues in the
tertiary structure of gp96.
Relating steady state anisotropy with lifetime (the Perrin equation
(Equation 3)) suggested that the rotational correlation time (
) of
the bound peptide-pyr (68.64 ns) increased relative to the unbound
state (27.64 ns) in low salt. This is presumably because the peptide in
the gp96-binding pocket is held in a somewhat restrained environment.
However, the rotational freedom (
) of the bound peptide appears to
depend on the solvent condition. The
value (68.64 ns) is greater in
low salt than in high salt (24.78 ns). This result fits well with our
earlier prediction that two bound peptides are brought close to each
other in a hydrophobic environment via gp96 dimer formation. The lower
values for bound pyr in high salt presumably means that the peptide
has greater rotational freedom in a more polar environment. The
anisotropy/lifetime values may be for a mixture of excimeric states in
low salt complexes. We surmise that the decrease in excited state
lifetime of the bound pyrene in high salt is probably related to its
increased hydrophilic environment.
 |
ACKNOWLEDGEMENTS |
We are extremely grateful to Dr. Pramod K. Srivastava and Sreyashi Basu (University of Connecticut, Farmington,
CT) for generously supplying mouse gp96, VSV peptide, and the bacterial
strain with plasmid and for helpful suggestions and discussions during
the course of this work. We thank Prof. Joshua Lederberg for his keen interest in the project, Dr. Thomas Sakmar and Ethan Marin for the use
of the spectrofluorimeter, Dr. Stephen K. Burley for the use of the
dynamic light scattering instrument, Dr John Kuriyan for making
available the mass spectrometer, and Dr. Brian T. Chait for help with
mass spectrometry.
 |
FOOTNOTES |
*
This work was supported by a grant from Antigenics LLC (New
York, NY). Additional support was provided by an instrumentation grant
from Hewlett-Packard Co. (Palo Alto, CA).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Molecular Genetics, Box 174, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8987; Fax: 212-327-8651; E-mail: sastrys{at}rockvax.rockefeller.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
HSP, heat shock
protein;
BA, butylamine;
HPLC, high performance liquid chromatography;
pyr, pyrene;
TEAA, triethylammonium acetate;
VSV, vasicular stomatitis
virus;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
MHC, major histocompatibility complex.
 |
REFERENCES |
-
Srivastava, P. K.,
and Heike, M.
(1991)
Semin. Immunol.
3,
57-64[Medline]
[Order article via Infotrieve]
-
Srivastava, P. K.,
Udono, H.,
Blachere, N. E.,
and Li, Z.
(1994)
Immunogenetics
39,
93-98[Medline]
[Order article via Infotrieve]
-
Suto, R.,
and Srivastava, P. K.
(1995)
Science
269,
1585-1588[Medline]
[Order article via Infotrieve]
-
Tamura, Y.,
Peng, P.,
Liu, K.,
Daou, M.,
and Srivastava, P. K.
(1997)
Science
278,
117-120[Abstract/Free Full Text]
-
Román, E.,
and Moreno, C.
(1996)
Immunology
88,
487-492[Medline]
[Order article via Infotrieve]
-
Ciupitu, A.-M.,
Peterson, M.,
O'Donnell, V.,
Williams, K.,
Jindal, S.,
Kiessling, R.,
and Welsh, R. M.
(1998)
J. Exp. Med.
187,
685-691[Abstract/Free Full Text]
-
Heikema, A.,
Agsteribbe, E.,
Wilschut, J.,
and Huckriede, A.
(1997)
Immunol. Lett.
57,
69-74[CrossRef][Medline]
[Order article via Infotrieve]
-
Srivastava, P. K.,
and Maki, R. G.
(1991)
Curr. Top. Microbiol. Immunol.
167,
109-123[Medline]
[Order article via Infotrieve]
-
Srivastava, P. K.
(1993)
Adv. Cancer Res.
62,
154-177
-
Udono, H.,
and Srivastava, P. K.
(1994)
J. Immunol.
152,
5398-5403[Abstract/Free Full Text]
-
Li, Z.,
and Srivastava, P. K.
(1993)
EMBO J.
12,
3143-3151[Abstract]
-
Blachere, N. E.,
Udono, H.,
Janetzki, S.,
Li, Z.,
Heike, M.,
and Srivastava, P. K.
(1993)
J. Immunotherapy
14,
352-356[Medline]
[Order article via Infotrieve]
-
Blachere, N. E.,
Li, Z.,
Chandawarkar, R. Y.,
Suto, R.,
Jaikaria, N. S.,
Basu, S.,
Udono, H.,
and Srivastava, P. K.
(1997)
J. Exp. Med.
186,
1315-1322[Abstract/Free Full Text]
-
Nieland, T. J.,
Tan, M. C.,
Monne-van Muijen, M.,
Koning, F.,
Kruisbeek, A. M.,
and van Bleek, G. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
6135-6139[Abstract/Free Full Text]
-
Nicchitta, C. V.
(1998)
Curr. Opinion in Immunol.
10,
103-109[CrossRef][Medline]
[Order article via Infotrieve]
-
Przepiorka, D.,
and Srivastava, P. K.
(1998)
Mol. Med. Today
4,
478-484[CrossRef][Medline]
[Order article via Infotrieve]
-
Breloer, M.,
Marti, T.,
Fleischer, B.,
and von Bonin, A.
(1998)
Eur. J. Immunol.
28,
1016-1021[CrossRef][Medline]
[Order article via Infotrieve]
-
Spee, P.,
and Neefjes, J.
(1997)
Eur. J. Immunol.
27,
2441-2449[Medline]
[Order article via Infotrieve]
-
Marusina, K.,
Reid, G.,
Gabathuler, R.,
Jefferies, W.,
and Monaco, J. J.
(1997)
Biochemistry
36,
856-863[CrossRef][Medline]
[Order article via Infotrieve]
-
Arnold, D.,
Wahl, C.,
Faath, S.,
Rammensee, H. G.,
and Schild, H.
(1997)
J. Exp. Med.
186,
461-466[Abstract/Free Full Text]
-
Lammert, E.,
Arnold, D.,
Nijenhuis, M.,
Momburg, F.,
Hammerling, G. J.,
Brunner, J.,
Stevanovic, S.,
Rammensee, H. G.,
and Schild, H.
(1997)
Eur. J. Immunol.
27,
923-927[Medline]
[Order article via Infotrieve]
-
Anderson, S. L.,
Shen, T.,
Lou, J.,
Xing, L.,
Blachere, N. E.,
Srivastava, P. K.,
and Rubin, B. Y.
(1994)
J. Exp. Med.
180,
1565-1569[Abstract]
-
Udono, H.,
Levey, D. L.,
and Srivastava, P. K.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
3077-3081[Abstract]
-
Janetzki, S.,
Blachere, N. E.,
and Srivastava, P. K.
(1998)
J. Immunotherapy.
21,
269-276[Medline]
[Order article via Infotrieve]
-
Srivastava, P. K.
(1997)
Methods
12,
165-171[CrossRef][Medline]
[Order article via Infotrieve]
-
Wearsch, P. A.,
and Nicchitta, C. V.
(1997)
J. Biol. Chem.
272,
5152-5156[Abstract/Free Full Text]
-
Wearsch, P. A.,
Voglino, L.,
and Nicchitta, C. V.
(1998)
Biochemistry
37,
5709-5719[CrossRef][Medline]
[Order article via Infotrieve]
-
Lakowicz, J. R.
(1983)
Principles of Fluorescence Spectroscopy, pp. 75-91, 128-132, and 347-357, Plenum Press, New York
-
Flynn, G. C.,
Chappell, T. G.,
and Rothman, J. E.
(1989)
Science
245,
385-390[Medline]
[Order article via Infotrieve]
-
Haugland, R. A.
(1996)
Handbook of Fluorescent Probes and Research Chemicals, 6th Ed., p. 36, Molecular Probes, Eugene, OR
-
Knopp, J. A.,
and Weber, G.
(1969)
J. Biol. Chem
244,
6309-6315[Abstract/Free Full Text]
-
Barrantes, F. J.,
Sakmann, B.,
Bonner, R.,
Eibl, H.,
and Jovin, T. M.
(1975)
Proc. Natl. Acad. Sci. U. S. A.
72,
3097-3101[Abstract]
-
Gorovits, B. M.,
and Horowitz, P. M.
(1995)
J. Biol. Chem.
270,
13057-13062[Abstract/Free Full Text]
-
Feng, L.,
Kim, E.,
Lee, W. L.,
Miller, C. J.,
Kuang, B.,
Reisler, E.,
and Rubenstein, P. A.
(1997)
J. Biol. Chem.
272,
16829-16837[Abstract/Free Full Text]
-
Winnik, F. M.
(1993)
Chem. Rev.
93,
587-614
-
Lehrer, S. S.
(1995)
Subcell. Biochem.
24,
115-132[Medline]
[Order article via Infotrieve]
-
Turro, N. J.
(1991)
Modern Molecular Photochemistry, pp. 141-143, University Science Books, Mill Valley, CA
-
Lehrer, S. S.
(1997)
Methods Enzymol.
278,
286-295[Medline]
[Order article via Infotrieve]
-
Egusa, S.,
Sisido, M.,
and Imanishi, Y.
(1985)
Macromolecules
18,
882-889
-
Forster, T.
(1948)
Ann. Phys.
2,
55-75
-
Engelke, M.,
Behmann, T.,
Ojeda, F.,
and Diehl, H. A.
(1994)
Chem. Phys. Lipids
72,
35-40[Medline]
[Order article via Infotrieve]
-
Engelke, M.,
Bojarski, P.,
Diehl, H. A.,
and Kubicki, A.
(1996)
J. Membr. Biol.
153,
117-123[CrossRef][Medline]
[Order article via Infotrieve]
-
Pap, E. H.,
van den Berg, P. A.,
Borst, J. W.,
and Visser, A. J.
(1995)
J. Biol. Chem.
270,
1254-1260[Abstract/Free Full Text]
-
von Hippel, P. H.,
and Schleich, T.
(1969)
in
The Structure and Stability of Biological Macromolecules (Timaschef, S. T., and Fasman, G. D., eds), pp. 417-574, Marcel Dekker, Inc., New York
-
Wearsch, P. A.,
and Nicchitta, C. V.
(1996)
Biochemistry
35,
16760-16769[CrossRef][Medline]
[Order article via Infotrieve]
-
Grosso, V. N.,
Previtali, C. M.,
and Chesta, C. A.
(1998)
Photochem. Photobiol.
68,
481-486
-
Srivastava, P. K.
(1994)
Experientia
50,
1054-1060[Medline]
[Order article via Infotrieve]
-
Creighton.
(1993)
Proteins: Structure and Molecular Properties, 2nd Ed., pp. 270-271, W. H. Freeman, New York
-
Boniface, J. J.,
Lyons, D. S.,
Wettstein, D. A.,
Allbritton, N. L.,
and Davis, M. M.
(1996)
J. Exp. Med.
183,
119-126[Abstract]
-
Stanfield, R. L.,
and Wilson, I. A.
(1995)
Curr. Opin. Struct. Biol.
5,
103-113[CrossRef][Medline]
[Order article via Infotrieve]
-
Weenink, S. M.,
and Gautam, A. M.
(1997)
Immunol. Cell Biol.
75,
69-81[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.