(Received for publication, October 10, 1995; and in revised form, December 5, 1995)
From the
An approach is described for the de novo design of
protein-like structures in which synthetic combinatorial libraries
(SCLs) were incorporated into an amphipathic -helical scaffold (an
18-mer sequence made up of leucine and lysine residues) to generate
conformationally defined SCLs. In particular, the SCLs in which the
``combinatorialized'' positions were on the hydrophilic face
showed an
-helical conformation in mild buffer. These SCLs were
used to generate context-independent but position-dependent scales of
-helical propensity for the L-amino acids. These scales were then
used to design highly
-helical peptides that self-associated in
mild buffer. The same approach was also found to permit the
identification of conformation-dependent decarboxylation catalysts.
Synthetic combinatorial libraries (SCLs) ()are
broadly recognized as having the capability of greatly accelerating the
discovery of new lead compounds. SCL approaches have primarily been
focused on the generation of small molecule diversities (i.e. short peptides, peptidomimetics, or organic
compounds)(1) . The generation of molecular diversities based
on defined structural motifs can be expected to broaden the use of SCLs
for those applications requiring the presence of a well defined
secondary and/or tertiary structure. For example, the de novo design of artificial receptors and catalysts in most cases
requires the generation of protein-like molecules having the general
structural and functional properties found in natural enzymes. Thus, a
productive strategy can be seen in the selection-based design of well
defined secondary and tertiary structures, which maintain sufficient
flexibility to allow the accessibility of the functionalities required
for catalysis to occur.
In an initial effort directed toward the
design of new protein-like structures using SCL approaches,
conformationally defined combinatorial libraries have been designed by
randomizing five positions in two different 26-amino acid-long
naturally occurring peptides. The first of these libraries, built in
our laboratory, was based on the amphipathic peptide
melittin(2) , whereas a CysHis
consensus ``zinc finger'' motif was used as a scaffold
by Bianchi et al.(3) . These first examples have
proven to be useful case studies to confirm that synthetic
combinatorial chemistry can be readily extended to structurally defined
polypeptides or protein segments.
Based on our earlier studies on
structure-activity relationships using basic
polypeptides(4, 5, 6) , a series of
conformationally defined SCLs were then constructed around an 18-mer
peptide composed solely of leucine and lysine residues(7) .
Leucine/lysine-based peptides represent simple model systems capable of
forming protein-like conformations through self-aggregation. The
resulting structures, however, lack the specific electrostatic,
hydrogen-bonded, and van der Waals interactions that are necessary to
stabilize unique protein conformations(8) . Therefore, we used
combinatorial diversity with the aim of overcoming a number of these
limitations while also allowing the design of new, functionalized,
positively charged, self-assembling proteins having unique, well
defined structures for specific functions. In an initial step, the
structural nature of these libraries was used to study the helical
propensity of amino acids, which permitted the identification of
peptides that self-aggregated into -helical conformations in mild
buffer.
The advantage of self-aggregated amphipathic peptides is their inherent tendency to produce hydrophobic cores, much like the intrinsic character of protein interiors, while providing an outwardly directed polar environment. Such characteristics have been applied in different studies for the design of new macromolecules that are able to specifically bind to ligands and/or to bring into close proximity functional groups that result in known biological functions(9, 10, 11, 12) . The conformationally defined SCL approach was used in the present study to design new protein-like structures that have binding step-based catalytic activity. The catalytic properties of individual self-aggregated peptides derived from these conformationally defined SCLs were explored with the decarboxylation of oxaloacetate serving as a model reaction system.
The GdnHCl denaturation studies were carried out by preparing mixtures of a stock solution of peptide mixture in buffer (5 mM MOPS-NaOH, 200 mM NaCl, pH 7.0), buffer alone, or a solution of 7.2 M GdnHCl in buffer. The ratios of buffer and 7.2 M GdnHCl solutions were varied to give the appropriate final GdnHCl concentrations. The samples were allowed to equilibrate for 30 min at room temperature prior to CD measurement.
Figure 1: Schematic representation of conformationally defined SCLs. The open circles represent the defined and mixture positions. The hydrophilic face is represented in black and the hydrophobic face in gray.
In order to
analyze the structural behavior of these three different libraries, the
apparent helical contents were measured for each peptide mixture using
CD spectroscopy either in MOPS buffer at neutral pH or in the presence
of 80% trifluoroethanol. In buffer, the CD spectra showed clear
indications of an -helical conformation only when the randomized
region was contained on the hydrophilic face of the helix (Fig. 2). The highest apparent ellipticity at 222 nm (negative
minimum characteristic for
-helices) was found when glutamic acid
was in the defined position (i.e. replacing lysine 6), and the
lowest apparent ellipticity occurred with proline in this position. In
addition, the helical content was found to be concentration-dependent,
indicating the formation of aggregates under these conditions. A
tetrameric aggregate was found to best fit the concentration dependence
curve obtained for the peptide mixtures tested in mild buffer (data not
shown). It should be noted that high ionic strength and high peptide
concentrations were necessary for the original YLK peptide to form
tetrameric aggregates. All of the peptide mixtures from the three
libraries were found to adopt an
-helical conformation in
trifluoroethanol with relatively small variations in apparent helical
content.
Figure 2: CD spectra of representative mixtures from the SCLs built on the hydrophobic face (A), the hydrophilic face (B), and the helix turn (C). The CD spectra were recorded in 5 mM MOPS buffer, 200 mM NaCl, pH 7, at 25 °C at a peptide mixture concentration of 150 µM. The spectra are shown for the peptide mixtures having at their defined positions Glu (solid line), Phe (dashed line), and Pro (centered line).
To properly mimic naturally occurring proteins, the de novo designed proteins should have a well defined conformation under physiological conditions. The library having its diversity on the hydrophilic face of YLK appeared to be more suitable for studies directed toward the generation of such proteins. Furthermore, the variation seen in apparent helical content between the peptide mixtures within this library is anticipated to allow the modulation of the helical content of final individual basic polypeptides. In order to obtain information on all of the randomized positions in a single screening process, this library was extended to generate four related libraries in which the O position was at position 6, 9, 13, or 16 (these four libraries represent a positional scanning SCL) ( Table 1and (20) ).
Both hydrogen bonding between the residues at positions i and i+4 or i-4 and the side chain interactions between residues at positions i and i+3 or i-3 are known to be the main contributors to helical stability. As summarized in Table 2, the extent of local random environments depends on the position of the defined amino acid within the sequence. For instance, a close to completely random local environment was achieved when the defined residue was located at position 9 or 13. In both cases, a random environment was provided by one of the two hydrogen bonds (on the carbonyl side or amide hydrogen side, respectively) and by the i-3 or i+3 position, respectively. In contrast, when the defined residue was located at position 6 or 16, a random environment was provided only by the position i+3 or i-3, respectively. A complete random environment having X positions at i±4 and i±3 could not be achieved while maintaining the amphipathicity (i.e. without substituting leucine residues) and/or maintaining the mixture positions in the middle of the hydrophilic face (i.e. at positions that are exposed to the solvent and distant from the hydrophobic/hydrophilic interface).
Each peptide mixture
from the four libraries was analyzed by CD spectroscopy in the absence
or the presence of GdnHCl. The stabilities of the peptide mixtures to
GdnHCl denaturation were measured by monitoring the mean residue
ellipticity at 222 nm as a function of denaturant concentration. In a
manner similar to proteins or single sequence defined polypeptides, the
mixtures unfolded cooperatively with midpoints that depended markedly
on the nature and position of the defined amino acid (Fig. 3).
The apparent helical content of the peptide mixtures defined with a
given amino acid was found to vary according to the location of this
amino acid within the sequence (i.e. which library the
corresponding peptide mixture belongs to). The degree of variability
was found to be amino acid-dependent. For instance, alanine at position
13 (in the middle of the helix) led to a higher helical content than
when it was located close to the N terminus (i.e. at position
6) (Fig. 4A). In contrast, a glycine at position 6 led
to a higher helical content when located near the N terminus than when
located in the middle of the helix or close to the C terminus (Fig. 4B). Other amino acids, such as asparagine, could
be located at any position without significant variations in the
resulting helical content (Fig. 4C). The apparent free
energy of helix formation (G
)
was calculated for each peptide mixture based on a tetramer formation,
assuming that the helix-coil transition is a totally cooperative
two-state transition(5, 30, 31) . The
resulting rank order of free energies for helix stabilization of each
amino acid by position in the sequence, (i.e. in a given local
environment) is summarized in Table 3. Similar values were
obtained when the free energy for helix formation was estimated from
the data resulting from GdnHCl denaturation and extrapolation of the
free energy at each individual concentration of GdnHCl back to zero
concentration (data not shown). In addition to the position dependence
found for the
G
values for
given amino acids, the relative rank orders of the amino acids varied
from one scale to the other. As expected, the highest similarity was
found for the scales generated from libraries having defined positions
at either 9 or 13, both of which represent a close to random local
environment for the amino acids studied. As in other scales presented
in the literature, proline was found to have the least helical
propensity, a property that was independent of its location. Proline
G
was 3-4 Kcal/mol lower
than the amino acid with the highest helical propensity in each scale.
Figure 3:
Guanidinium hydrochloride denaturation
curves. The GdnHCl denaturation curves were determined for the peptide
mixtures having a defined Ala (), Leu (
), and Gly (
)
at position 13. The [
]
values were
measured at various GdnHCl concentrations with a peptide mixture
concentration of 160 µM in 5 mM MOPS buffer, 200
mM NaCl, pH 7, at 25 °C.
Figure 4: CD spectra of peptide mixtures from the SCLs defined with alanine (A), glycine (B), or asparagine (C). The CD spectra were recorded as described in Fig. 2. The spectra are shown for the defined residue at position 6 (solid line), 9 (dotted line), 13 ( dashed line), and 16 ( centered line).
The discrepancy seen between the scales generated from the libraries
representing ``fixed'' local environments (defined position
at 6 or 16) and those representing ``random'' local
environments (defined position at 9 or 13) further confirms that the
nature of amino acid-amino acid interaction plays an important role in
determining the helical propensity. We therefore believe that a local
random environment can translate the average helical propensity of an
amino acid, which, in turn, can be expected to assist in understanding
the secondary structure of a given peptide sequence. In addition, these
results show that conformationally defined SCLs providing random local
environments can be used to yield an overall helical propensity value
for each amino acid that is independent of the nature of the
interacting amino acid side chains and of the contribution of the
donor/acceptor pair of hydrogen bonding amino acids. When comparing the
relative free energies of helix formation obtained in a close to random
environment with other experimental and statistical data, the best
linear correlation was with the relative free energies required to
orient the amino acids in helical dihedral angles described by
Muñoz and Serrano (29) (Fig. 5).
This can be explained by the fact that in contrast to other approaches,
this method does not involve the contribution of the hydrogen bonds and
side chain-side chain interactions. It should be noted that the
relative effect toward the stability of the -helix by the six
amino acids Ala, Ile, Met, Gln, Val, and Thr correlate well for all the
different proposed
scales(21, 22, 23, 24, 25, 26, 27, 28, 29) .
Thus, these amino acids can be considered as helix stabilizers or
destabilizers regardless of their local environment. The analysis of
the relative effect of the other 14 naturally occurring amino acids
shows significant discrepancies between the different scales,
indicating their greater susceptibility to their local environment.
Figure 5:
Correlation between
G
and
G
. The
G
represents the
G
of each amino acid when
defined at position 13 relative to the
G
of glycine and was divided by
four to account for the contribution of the monomeric units. The
G
represents the intrinsic
G of
each amino acid relative to the intrinsic
G of glycine reported by
Muñoz and Serrano(29) . The correlation
coefficient of these values (after removing the values for Arg, Leu,
Ser, and Trp) was 0.91, and the slope was
0.95.
In order to test whether the average positional helical propensity
found in the present studies can be translated to individual peptides,
three different series of peptides were prepared. In two of the series,
the peptides represented the combination at positions 6, 9, 13, and 16
of either the four amino acids with the highest or, separately, the
lowest helical propensity from each library or combinations of amino
acids having high propensity with others having low helical propensity.
The first series of peptides was based on the original YLK peptide,
whereas the second series was synthesized incorporating these amino
acids into an intrinsically less amphipathic 18-mer peptide composed
solely of alanine and serine residues (termed YAS) (Table 4). In
agreement with our scales, the peptide
YLK[WL
A
E
]
showed the highest helical content in aqueous buffer when compared with
the other peptides of its series (Table 4). Similarly, in aqueous
buffer, the
-helical content in the [YAS] series was
qualitatively found to be
YAS[W
L
A
E
]
>
YAS[W
L
R
I
]
>
YAS[N
K
A
E
]
>
YAS[L
P
P
P
] (Table 4). A completely unrelated peptide, PGLa, was also
selected for validation of the described scales. PGLa is a 21-residue
naturally occurring peptide isolated from frog skin that is known to be
inducible into an amphipathic
-helical conformation(32) .
Two PGLa analogs were prepared by replacing the residues at positions
6, 9, 13, and 16 either with the four amino acids having the highest
helical propensity in the respective positional scales (i.e. Trp, Leu, Ala, and Glu, respectively) or with four alanine
residues, because alanine shows one of the highest helical propensity
in most of the reported scales (21, 22, 23, 24, 25, 26, 27, 28, 29) .
Although the helical content was low for the three peptides in MOPS
buffer, analog
PGLa[W
L
A
E
]
had the highest helical content, followed by
PGLa[A
A
A
A
]
and PGLa (Table 4). Greater differences in ellipticities were
observed in 10% trifluoroethanol, which allows the detection of weak
helical tendencies in unstructured peptides ([
]
= -7793, -5377, and -4331 deg cm
dmol
for
PGLa[W
L
A
E
],
PGLa[A
A
A
A
],
and PGLa, respectively).
The similarities in the helical content
rank order found between these three separate series illustrate the
potential of average helical propensity in the design of new,
-helical basic polypeptides. In particular, using conformationally
defined SCLs, we were able to modify in a single optimization step the
sequence of the original YLK scaffold to generate individual sequences
having well defined secondary structures in mild buffer. Furthermore,
the use of the nonsequence-related PGLa series shows that one can
increase the helical content of a given sequence by incorporating high
propensity amino acids as determined using random local environment
scales at specific positions. Overall, these results support the
contribution positional dependence makes to the
-helical stability
of amino acids.
Each peptide
mixture was assayed for its ability to enhance the decarboxylation of
oxaloacetate. The original YLK sequence and oxaldie-1 served as
references in the assay used in this study and showed similar specific
activities. All of the peptide mixtures, except when proline was at the
defined position, were found to have equal or higher activity when
compared with the original YLK and oxaldie-1. The good correlations
observed between the specific activity and the ability of the mixtures
to adopt an -helical conformation in buffer (Fig. 6)
support the reported role of this conformation in the catalytic
activity of such peptides. This is confirmed by the fact that none of
the peptide mixtures from the library built on the hydrophobic face of
the helix showed significant activity. It should also be noted that
none of these later peptide mixtures adopted a defined conformation in
buffer (Fig. 2A).
Figure 6:
Correlation between decarboxylation
activity and the apparent ellipticity at 222 nm of the SCLs. The
specific activities () were determined at a peptide mixture
concentration of 0.2 mM with 11.4 mM oxaloacetate in
phosphate-buffered saline buffer at 25 °C. The
[
]
values (
) were measured at a
peptide mixture concentration of 160 µM in 5 mM MOPS buffer, 200 mM NaCl, pH 7, at 25 °C. Each graph
represents a single positional SCL, and the x axis represents
the defined amino acids.
The identification of active
individual sequences from the screening of an SCL in a positional
scanning format involves selecting the most active peptide mixtures
from each individual library and using combinations of the
corresponding defined amino acids to prepare all of the possible
individual peptides(20) . Thus, a set of individual peptides
was prepared that represented all possible combinations of the amino
acid(s) selected at each position from the screening assay: Glu, Lys,
or Trp at position 6; Ala, Asn, or Trp at position 9; Glu, Gln, Val, or
Trp at position 13; and Asp, Met, Gln, or Arg at position 16. The
combinations of these amino acids resulted in the generation of a set
of 144 individual 18-mer peptides (3 3
4
4
= 144). Each individual peptide was screened for catalytic
activity in a manner similar to that carried out for the libraries. The
specific activities ranged from 10.2
10
to
3.9
10
s
(representative
sequences are shown in Table 5). The most active individual
peptides were found to catalyze the decarboxylation of oxaloacetate via
a UV-detectable enamine intermediate (11, 33) with
Michaelis-Menten saturation kinetics corresponding to a K
value 3-4-fold higher than that found
for oxaldie-1 under the assay conditions used (Fig. 7). This
activity is in the same order of magnitude as that first reported for
catalytic antibodies (34) and represents a
10
-10
-fold enhancement over the same
reaction catalyzed by simple amines(11) . The activity found
for these peptides was related to their ability to fold into an
-helical conformation in buffer ([
]
= -28300 and -4460 deg cm
dmol
for YKLLKELLAKLKWLLRKL-NH
and
oxaldie-1, respectively; peptide concentration was 200 µM in 5 mM MOPS buffer, 200 mM NaCl, pH 7, 25
°C). A negative control peptide containing three proline residues
was included in this set of peptides in order to confirm the low
activity observed for the peptide mixtures defined with proline. As
anticipated, this peptide had lower activity than any of the 144
peptides tested (specific activity of 3.75 10
s
), combined with a low helical content in
buffer ([
]
= -3985 deg
cm
dmol
).
Figure 7:
Kinetic analysis for the decarboxylation
activity of individual peptides derived from the SCLs. All the
parameters were determined in phosphate-buffered saline buffer at 25
°C. The peptide concentration was selected at 0.2 mM,
which was found to be in the linear section of the maximum rate versus peptide concentration plots. The initial rates are
plotted for YKLLKELLAKLKWLLRKL-NH (
),
YKLLKLLLPKLKPLLPKL-NH
(
), oxaldie-1 (
), and
spontaneous hydrolysis (
). The derived kinetic parameters are
shown in the top panel.