(Received for publication, February 10, 1995; and in revised form, April 10, 1995)
From the
Antigenic peptides are translocated into the lumen of the
endoplasmic reticulum by the action of the transporter associated with
antigen processing (TAP), where they are subsequently needed for the
correct assembly of major histocompatability complex molecules. The
transport function was reconstituted in insect cells by expression of
both TAP genes. On the basis of this overexpression system, substrate
selection was analyzed in detail by a direct bimolecular peptide
binding assay. Competition assays with peptide variants, including
substitutions of residues with alanine or structurally related amino
acids, underline the broad peptide specificity of the human TAP
complex. Steric requirements of the substrate-binding pocket were
mapped using elongated peptides and scans with bulky, hydrophobic amino
acids. Complex nonapeptide libraries were used to determine the
contribution of each residue to stabilize peptide-TAP complexes. For
the first time, this approach lets us directly evaluate the importance
of peptide selection for the overall process of antigen presentation on
the level of the peptide transporter.
Cytotoxic T lymphocytes recognize peptides derived from
endogenous proteins in association with major histocompatibility
complex (MHC) ( Members of this ATP-binding cassette superfamily,
including the multidrug resistance P-glycoprotein and the oligopeptide
transporter of Salmonella typhimurium, are involved in the
transport of substrates across membranes (Higgins, 1992).
Hydrophobicity profiles of these proteins suggest two domains of six to
eight membrane-spanning regions and two hydrophilic domains that
contain the ATP binding motifs Walker A and Walker B. Indeed, it was
shown by ATP cross-linking that the individually expressed hydrophilic
C-terminal domains of either TAP1 or TAP2 bind ATP
(Mller et al., 1994; Wang et
al., 1994). Several assay systems have been developed so far to
confirm the function of the TAP complex as peptide translocator. These
experiments are based on the restoration of cytotoxic T cell
recognition (i.e. correct antigen presentation in mutant cell
lines by transfection with tap genes (Spies & DeMars,
1991)) or accumulation of peptides in the ER of permeabilized cells or
TAP1 In order to circumvent the
problems associated with these indirect assays of peptide
translocation, a direct bimolecular binding and competition assay was
established, because an isolated and reconstituted in vitro translocation system is not available so far. Substrate
specificity and selection of the TAP complex were examined in detail by
competition studies using reporter and various modified competitor
peptides. Although peptide binding was discussed in studies relying on
microsome-associated radioactivity, peptide binding to TAP1
Synthetic nonapeptide libraries were generated on a robot
system using Fmoc amino acids, solid phase chemistry, and Fmoc
leucine-loaded Wang resin or an equimolar mixture of 19 Fmoc amino acid
resins loaded with all proteinogenic amino acids except cysteine.
Equimolar mixtures of Fmoc amino acids equimolar to the coupling sites
on the resins were used for the X positions, and 5-fold molar
excess was used for coupling of single amino acids in defined
positions, using the diisopropylcarbodiimide/1-hydroxybenzotriazole
method for coupling. Details of the procedure are described elsewhere
(Udaka et al., 1995). To confirm the sequences and the amino
acid frequency in the X positions, the peptide library was
subjected to amino acid analysis, pool sequence analysis (Stevanovic
& Jung, 1993), and electrospray mass spectrometry (Metzger et
al., 1994). Close to equimolar distribution of every amino acid
was found to be within the error limits of the analytical methods
(Kienle et al., 1994). Peptide concentrations were determined
using an orthophthalic aldehyde assay (Pierce).
Figure 1:
Peptide binding
affinity of the reporter peptide *RRYNASTEL* used in the experiments.
Each value (
Figure 2:
Summary of competition assays using
peptides with alanine and homologous substitutions of RRYNASTEL. In
relation to RRYNASTEL (IC
Figure 3:
Summary of competition assays using
elongated peptides of the modified epitope of histone H3 (RRYNASTEL).
The IC
Figure 4:
Summary of competition assays using
peptide scans containing bulky, hydrophobic amino acids of
(1-naphthyl)alanine and
In an inverse
competition experiment, a 14-fold molar excess of unlabeled RRYNASTEL
as competitor was needed to achieve 50% inhibition of binding of the
dansylated, radiolabeled peptide, *RRYUASTEL* (data not shown). An
IC
Figure 5:
ATP- and TAP-dependent translocation of
the
In Fig. 6, the IC
Figure 6:
Competition assays using complex peptide
libraries. To analyze the effect of each amino acid residue on peptide
selection, one position of the peptide epitope RRYNSATEL was kept
constant and the rest was randomized by 19 amino acids (with the
exception of cysteine). The IC
Peptide binding to microsomes at 4 °C was found to be
highly TAP1 Peptide binding studies allow us to determine
substrate specificity of the TAP complex without the limitations of the
translocation assays due to the absence of a retention system, peptide
degradation, or an export machinery that may represent a bias for
certain peptides. The fit of our data to the theoretical saturation
binding function in the standard and Scatchard plot implies that TAP
binding obeys a typical ligand-receptor interaction with identical,
independent binding sites. In consequence, this allows us to use the
competition assay for the determination of the IC In detail, the experiments with elongated
peptides point to a stabilizing effect of peptide binding by a
N-terminal arginine. This is strongly supported by the evaluation of
the stabilization effect determined individually for each residue with
the use of random peptide libraries, which shows that the strongest
contribution of Most
strikingly, the binding affinity of peptides with bulky, hydrophobic
amino acids, such as (1-naphthyl)alanine or The use of complex peptide libraries provides further clues
on the function of the TAP complex. The relatively broad peptide
specificity of human TAP (Androlewicz et al., 1993; Momburg et al., 1994; Androlewicz & Cresswell, 1994), which is
reflected in the variety of MHC class I bound peptides, was confirmed
by the fact that the affinity of the reporter peptide RRYNASTEL is only
11 times higher than the affinity of the totally random nonapeptide
library (X For
the first time, the restrictions of TAP on the repertoire of peptides
available for presentation on MHC class I molecules can be directly
quantified. The IC
The single letter code is used for peptide sequences. In addition, B
means (1-naphthyl)alanine and U
)class I molecules. These peptides are
believed to be translocated into the lumen of the endoplasmic reticulum
(ER) by the action of the transporter associated with antigen
processing (TAP) for binding to assembling MHC class I molecules
(Neefjes et al., 1993; Shepherd et al., 1993;
Androlewicz et al., 1993). Formation of MHC-peptide complexes
induces a conformational change that is required to overcome retention
in the ER and to allow subsequent transport of these complexes to the
cell surface (Suh et al., 1994; Ortmann et al.,
1994). The transport complex is a heteromer (Spies et al.,
1992; Kelly et al., 1992; Meyer et al., 1994)
composed of two MHC-encoded subunits, TAP1 and TAP2, with homology to
the ATP-binding cassette superfamily of transport proteins (Monaco, et al., 1990). The stoichiometry of the solubilized and
purified TAP complex was revealed to be 1:1 (TAP1/TAP2) (Meyer et
al., 1994).
TAP2-containing microsomes measured by quantitation of
radiolabeled peptide. The latter can be recovered as peptide
glycosylated in the ER (Neefjes et al., 1993), or microsomal
lumen (Meyer et al., 1994), or as peptide bound to MHC class I
molecules (Shepherd et al., 1993; Androlewicz et al.,
1993) or simply associated with microsomes (Shepherd et al.,
1993; Heemels et al., 1993; van Endert et al., 1994).
Translocation was demonstrated to be ATP-dependent. Peptide specificity (e.g. C-terminal preference and length selectivity) was
examined in these and various other studies (Schumacher et
al., 1994a, 1994b; Momburg et al., 1994; Androlewicz
& Cresswell, 1994). Nevertheless, all of these previous assay
systems are based on a retention system (i.e. glycosylation or
MHC class I binding), because transported peptides without it were hard
to detect, possibly because of another active export system for
peptides (Schumacher et al., 1994a). It remains to be shown
that an assumed substrate specificity of the retention system, a
proteolytic activity in the cytoplasm and ER, or the export machinery
do not alter the experimental results.
TAP2
has recently been shown by photoaffinity cross-linking of peptides
(Androlewicz & Cresswell, 1994; Androlewicz et al., 1994)
and binding assays involving an overexpression system for human
TAP1
TAP2 in insect cells, which has been used to screen various
peptides derived from MHC class I epitopes (Meyer et al.,
1994; van Endert et al., 1994). In this article, we report a
systematic approach to determine the requirements for peptide-TAP
complex formation, using variants of the histone H3-derived peptide
(RRYNASTEL) that have been elongated or modified by alanine,
structurally related, or bulky amino acids. In addition, complex
nonapeptide libraries, including all possible sequence variations, are
used to determine the stabilization or destabilization effect for each
peptide residue individually in terms of Gibbs free enthalpy
(
G).
Overexpression of TAP1
The generation of recombinant
baculoviruses carrying genes of human tap1 and tap2 has been described previously (Meyer, et al., 1994). Sf9 (Spodoptera frugiperda) cells were grown as a monolayer
according to standard procedures in TC100 insect cell medium with 10%
fetal calf serum, and infection was routinely performed with a
multiplicity of infection of 3-5.TAP2 with the
Baculovirus Expression System
Preparation of Microsomes
Sf9 cells were harvested
60-72 h postinfection by centrifugation and washed once with
phosphate-buffered saline. The cells were lysed after resuspension in
cavitation buffer (250 mM sucrose, 25 mM KOAc, 5
mM MgOAc, 0.5 mM CaOAc, 50 mM Tris-Cl, pH
7.4) with proteinase inhibitor mix at 10 cells/ml by
repeated drawing through a 26 gauge needle. Nuclei and nonlysed cells
were removed by centrifugation (300
g for 5 min at 4
°C), and the supernatant was diluted 6.4-fold with 2.5 M
sucrose in gradient buffer (150 mM KOAc, 5 mM MgOAc,
50 mM Tris-Cl, pH 7.4) and stepwise overlaid with 2.0 and 1.3 M sucrose in gradient buffer and with cavitation buffer. After
centrifugation overnight (104,000
g at 4 °C), the
turbid fraction at the interface of the 2.0 and 1.3 M sucrose
solution was collected, diluted 2-fold with phosphate-buffered saline
and 1 mM 1,4-dithio-DL-threitol and centrifuged
(227,000
g for 1 h at 4 °C). The vesicles were
resuspended in phosphate-buffered saline and 1 mM
1,4-dithio-DL-threitol, snap frozen in liquid nitrogen, and
stored at -80 °C.
Synthesis of Peptides
The peptides in this study
were synthesized on a multiple peptide synthesizer (MultiSynTech,
Bochum, Germany) by conventional Fmoc chemistry. Identity of the
peptides was checked by mass spectrometry, and the purity was
determined by reversed phase HPLC. Purity was at least 93% for each
peptide of the alanine scan, 89% for the
-(1-naphthyl)-L-alanine scan, and 80% for the other
peptides. The reporter peptide (RRYNASTEL) was HPLC-purified using a
C-18 column and a gradient of acetonitrile in water (purity, >99%).
Peptides containing
-dansyl-lysine were synthesized by removal of
the Boc group from N-
-Z-N-
-Boc-Lys-OH with
40% trifluoroacetic acid in water, reaction of the product with
dansylchloride in acetone/NaOH, removal of the Z group by catalytic
hydrogenation, reaction with Fmoc-N-hydroxysuccinimide, and
subsequent use of the product after crystallization in peptide
synthesis.
Iodination of Peptides
Peptides were iodinated as
described previously (Hunter & Greenwood, 1962). In brief, the
reaction was routinely performed with 15 nmol of peptide and 3.7
10
Bq (1 mCi) Na
I using the
chloramine-T method. Free iodine was removed by gel filtration through
a Sephadex G-10 column (Pharmacia). The specific activities were
0.8-1.3
10
Bq/mol for *RRYNASTEL* and
0.2-0.3
10
Bq/mol for
-dansyl-lysine-containing peptides.
Peptide Binding and Competition Assays
Microsomes
were diluted with assay buffer (phosphate-buffered saline with 1 mg/ml
dialyzed bovine serum albumin, 1 mM
1,4-dithio-DL-threitol, 2 mM MgCl) to a
final protein concentration of 75 µg/ml in binding assays and
25-50 µg/ml in competition assays. The protein concentration
was determined using the bicinchoninic acid assay (Pierce). This
suspension was homogenized by drawing through a 23 gauge needle. For
the determination of the binding affinity of TAP1
TAP2, various
amounts of radiolabeled reporter peptide *RRYNASTEL* were added as
indicated at 4 °C. In the case of competition assays, 300 nM of the reporter peptide and the appropriate amount of unlabeled
competitor were added at 4 °C. After incubation for 5 min on ice,
350 µl of ice-cold assay buffer were added, and the microsomes were
pelleted by centrifugation (12,000
g for 10 min at 4
°C). Vesicle-associated radioactivity was quantified by
counting after one washing step with 500 µl of ice-cold assay
buffer. The amount of bound peptide was corrected by unspecific binding
of baculovirus wild type microsomes. The data set was fitted by the
competition function: the percentage of inhibition = 100
([competitor]/IC
)/(([competitor]/IC
)
+ 1)). The concentration needed for 50% inhibition was determined
from three to four competition assays.
Peptide Translocation Assays
Peptide translocation
assays were performed as described previously (Meyer et al.,
1994). In brief, 150 µl of microsome suspension (20 µg of total
amount of protein) were incubated with 50 ng of radioactive peptide in
the presence of 0.3 unit of apyrase, 10 mM ATP, 100-fold
excess of competitor peptide, or nonhydrolyzable ATP analogue (10
mM AMP-PNP). After 10 min of incubation at 37 °C,
microsomes were lysed by adding detergent. Glycosylated and therefore
translocated peptides are quantified by counting after binding to
concanavalin A-Sepharose (Sigma) and elution with
-methylmannoside.
Peptide Binding to the TAP Complex
The affinity
constant of the radiolabeled peptide, *RRYNASTEL*, for the TAP complex
was determined in a saturation binding experiment to be 310 ± 30
nM at 4 °C (Fig. 1). No specific binding to
microsomes containing either only TAP1 or TAP2 was observed (data not
shown). The linear Scatchard plot indicates a noncooperative
ligand-receptor interaction with identical, independent binding sites.
Saturation was reached at 0.175 nmol/mg microsomal protein. Assuming
one binding site and a molecular mass of about 150 kDa of the transport
complex, 2.4% of the total microsomal protein represents functional TAP
complex expressed in insect cells. The affinity constant and the
saturation value are not influenced by ATP (data not shown). Peptide
binding to a high affinity substrate binding site generated by TAP1 and
TAP2 therefore represents an ATP-independent process, whereas peptide
translocation was shown before to be strictly ATP-dependent (Meyer et al., 1994).
) represents specific binding to
TAP1
TAP2-containing microsomes background-corrected for binding
to the equivalent amount of microsomes from baculovirus wild
type-infected insect cells (
). The total protein amount for
each assay (150 µl) was 11.5 µg. The inset shows the
data of a Scatchard plot leading to K
= 310 ± 30 nM. Saturation is reached
at 0.175 nmol/mg microsomal protein.
Amino Acid Substitutions
To analyze the affinity
of various peptides for the TAP complex, competition assays were
carried out using *RRYNASTEL* (300 nM) as reporter peptide.
The percentage of inhibition of peptide binding at various competitor
concentrations was plotted against the molar excess of competitor. The
concentration needed for 50% inhibition (IC) was
calculated by fitting a competition function to the data set. In order
to compare various peptides, the IC
of each competitor was
expressed in relation to the IC
of the reference peptide
(RRYNASTEL). To reveal possible anchoring positions for peptide
binding, each amino acid at an individual position of the reporter
peptide (RRYNASTEL) was sequentially exchanged against an alanine. The
results of this alanine scan and of the substitutions by related amino
acids are summarized in Fig. 2. With the exception of
phenylalanine at position 9 (IC
/IC
= 0.27), nearly all peptides show a decreased affinity for
TAP1
TAP2, ranging from an up to 10-fold lower affinity of
peptides with a changed N or C terminus to nearly unchanged affinity by
exchange of glutamic acid to alanine in position 8
(IC
/IC
= 0.88). The
destabilization effect of each lysine in position 1 and 2 is additive
with relation to the free enthalpy. The slightly increased affinity of
peptide substitution, asparagine to glutamine at position 4, reflects
the 2-fold higher affinity of TAP for the original histone H3-derived
epitope RRYQKSTEL compared with the reporter peptide RRYNASTEL that is
modified with a glycosylation sequence (Fig. 3).
), the
IC
/IC
values of these competitor
peptides are given. Varied positions in the peptides are underlined.
/IC
ratios of various competitor
peptides are given. For comparison, the data of the original epitope
(RRYQKSTEL) and the reference peptide (RRYNASTEL) of human histone H3
are given on top. Varied positions in the peptides are underlined.
Elongated Peptide Epitopes
Within the scope of
antigen processing, protein fragments variable in size are generated in
the cytosol or nucleus. In order to determine the length selectivity of
the TAP complex, elongated modifications derived from the histone H3
epitope were used (Fig. 3). No length selectivity was observed
in the range of 9-15-mers, because the longest peptide tested
(REIRRYNASTELLIR) showed only a slightly decreased binding affinity
(IC/IC
= 1.25). Interestingly,
peptides containing an N-terminal arginine bound very efficiently,
whereas peptides with an altered amino acid at position 1 have a
decreased binding affinity.
Bulky, Hydrophobic Substitutions
In experiments
originally designed to introduce cross-linker, biotin, or fluorescence
labels and to map the steric requirements of peptide binding to the TAP
complex, peptides that contain substitutions of (1-naphthyl)alanine or
-dansyl-lysine at different positions were screened for binding to
the TAP complex (Fig. 4). Most strikingly, almost every
substitution causes an increase in peptide affinity independent of the
relative position of the bulky, aromatic side chain in comparison with
the reference peptide and reached values of 12-fold increased affinity
for peptides dansylated at positions 6-8
(IC
/IC
= 0.081). Only the peptide
with an N-terminal (1-naphthyl)alanine exhibits a decreased affinity
(IC
/IC
= 11.3).
-dansyl-lysine. The IC
values (top) of these modified peptides are given in reference to the
IC
of RRYNASTEL along with the structures (bottom) of the modifications. Varied positions in the
peptides are underlined.
/IC
of >200 was observed for the
amino acid
-dansyl-lysine in an experiment to rule out the
possibility of an unspecific adsorption of these peptides to the
peptide binding pocket via the aromatic side chain. The addition of an
excess of lipid did not affect the IC
value of RRYUASTEL,
demonstrating that dansylated peptides do not accumulate in or at the
membrane, thereby leading to an increase of the peptide concentration
and the apparent binding constant. In the absence and the presence of a
20-fold excess of microsomal membranes (Sf9 baculovirus wild type) the
IC
values of the dansylated peptide (RRYUASTEL) are found
to be identical within the range of error (data not shown). We can
further demonstrate that the dansylated, radiolabeled peptide
(*RRYNASTUL*) is directly translocated in an ATP- and TAP-dependent
fashion (Fig. 5).
-dansyl-lysine-modified peptide *RRYNASTUL* at 37 °C.
Microsomes (20 µg of total amount of protein) were incubated with
0.3 unit of apyrase (- ATP), 10 mM ATP (+ ATP), 100-fold excess of unlabeled competitor peptide (RRYNASTUL)
and 10 mM ATP (+ competitor), or 10 mM AMP-PNP (+ AMP-PNP). Glycosylated and therefore
transported peptide was isolated by binding to concanavalin A-Sepharose
and elution with
-methylmannoside. ConA, concanavalin A; BVwt, baculovirus wild type.
Peptide Libraries
We investigated the stabilizing
or destabilizing effect of each individual peptide residue of the
epitope RRYNASTEL bound to TAP, using peptide libraries representing up
to 19 = 322,687,697,779 nonapeptides that are
present at almost the same frequency. Therefore, we synthesized a
nonapeptide library (X
) that contains every
proteinogenic amino acid except cysteine at every position at the same
frequency (X
) and sublibraries (X
O) containing one conserved residue (O) from
each position of the reference epitope (RRYNASTEL). The IC
of each sublibrary was determined in a competition assay using
RRYNASTEL as reporter peptide. Binding was titrated to background level
(100% inhibition), indicating that a large fraction out of the
nonapeptide library can compete for TAP binding. Interestingly, only an
11-fold excess of totally randomized nonapeptides (X
) is needed for 50% inhibition, reflecting the
broad substrate specificity of the human TAP complex.
values of each sublibrary (X
O) in relation to the IC
of the
totally randomized library (X
) were expressed in
values of
G, representing the stabilizing and destabilizing
effect of each individual amino acid residue. Three groups of residues
can be identified: (i) residues that promote peptide binding
(
G < -1.7 kJ/mol), (ii) residues with little effect
on the binding affinity, and (iii) residues that do not favor peptide
binding to TAP (
G > +1.7 kJ/mol). In agreement to the
alanine and homologous peptide substitutions, the first three residues
of RRYNASTEL were found to be optimized for binding to the TAP complex.
The strongest stabilization effect was observed with N-terminal
arginine with a
G = -4.6 kJ/mol.
= 11.0 of this X
peptide library was taken as reference. The
IC
/IC
of each sublibrary (X
O) are expressed in terms of
G,
representing a stabilizing (-
G) or destabilizing
effect (+
G) of an individual amino acid residue.
TAP2-specific and ATP-independent. The dissociation
constant was determined to be 310 ± 30 nM. This is
consistent with recent findings of peptide binding to TAP at low
temperatures (van Endert et al., 1994). Although there is a
strict requirement of ATP hydrolysis for peptide translocation mediated
by TAP (Neefjes et al., 1993; Shepherd et al., 1993,
Meyer et al., 1994), we assume a mechanism of translocation
that consists of at least two steps. If this mechanism involves
transition from a high peptide affinity state to a low affinity state
through binding of ATP (van Endert et al., 1994) or a
conformational change after ATP hydrolysis remains to be elucidated,
then our data indicate that addition of ATP does not alter the peptide
binding affinity nor the accessible binding sites of the TAP complex at
these temperatures.
values
for various peptides. Unlike MHC class I molecules, no anchoring
positions that are strictly required for peptide binding and that
drastically decrease binding affinity, if altered, could be identified
so far. We observe the strongest contribution to the binding affinity
from residues at or close to the N or C terminus but with the effect
being far from dramatic.
G comes from this residue. Although
acetylation (Schumacher et al., 1994a) and methylation
(Momburg et al., 1994) of peptides were reported to decrease
peptide translocation by TAP, no marked influence was reported so far
with respect to the N-terminal residue (Momburg et al., 1994).
With a C-terminal substitution to phenylalanine
(IC
/IC
= 0.27, Fig. 3) we
have identified a peptide consisting of proteinogenic amino acids that
has an increased affinity compared with the reporter peptide. Taking
into account the stabilizing effect of a C-terminal leucine, as
determined from the random peptide library, we find that the
hydrophobic residues leucine and phenylalanine are preferred to
alanine, a pattern that correlates with the substrate specificity
reported for the mouse TAP1
TAP2 but not with that found for human
TAP (Momburg et al., 1994). The missing length selectivity up
to a 15-mer is in line with recent findings that peptides of 8-16
amino acids are equally efficient in peptide competition (van Endert et al., 1994) and even a 24-mer efficiently competes for
peptide translocation (Androlewicz & Cresswell, 1994).
-dansyl-lysine is
significantly increased. This gives us the prospect of incorporating
other amino acids with bulky side chains, for example photoaffinity
cross-linkers, fluorescence, or biotin labels, to further investigate
the molecular architecture and function of the TAP complex. The fact
that these hydrophobic amino acids are preferred to naturally occurring
amino acids may point to an amphipathic character of the peptide
binding pocket formed by the N-terminal transmembrane domains of TAP1
and TAP2 at the lipid-protein interface or at amphipathic transmembrane
helices.
). The lack of a defined anchoring
residue, identifiable by comparison of the effects of individual
residues with the random library X
, as well as the
toleration of bulky amino acids at nearly every position, leads us to
the speculation that the strongest contribution to peptide binding
comes from the backbone and not through fixation of side chains.
/IC
of 11 for the
nonapeptide library X
compared with an 180-fold
increased binding affinity of the H-2K
-restricted epitope
SIINFEKL over an octapeptide library (X
) measured
through the ability to stabilize the conformation of peptide-depleted
MHC class I molecules (Udaka et al., 1995), points clearly to
a restrictive role for TAP. With regard to the different combinatorial
frequencies of each peptide in a 8- and 9-mer peptide library and the
already optimized MHC-binding of the epitope SIINFEKL, this difference
in the IC
values clearly represents an upper limit. In
conclusion, this means that the TAP1
TAP2 complex has a small but
significant effect on the overall process of selection of peptides for
antigen presentation.
-dansyl-lysine. X stands
for a mixed position in peptide libraries, where all proteinogenic
amino acids (except cysteine) are randomly incorporated and present at
the same frequency. O means one defined residue within the peptide
sublibraries.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.