Peptide self-assembly as a model of proteins in the
pre-genomic world
Indraneel Ghosh1 and Jean Chmielewski2
Excellent catalytic efficiency has been obtained within a
series of self replicating peptides, and nucleobase inclusion
into a salt-switchable self replicating peptide is found to
override the switch. Interestingly, cross-catalytic formation
of an RNA aptamer is reported with a cationic peptide,
and novel, amide-based biopolymers have been designed
to self assemble.
Addresses
1
Department of Chemistry, University of Arizona, 1306 E. University
Blvd, Tuscon, AZ 85721, USA
e-mail: [email protected]
2
Department of Chemistry, Purdue University, 560 Oval Drive,
West Lafayette, IN 47907, USA
e-mail: [email protected]
Current Opinion in Chemical Biology 2004, 8:640–644
This review comes from a themed section on
Model systems
Edited by David G Lynn and Nicholas V Hud
Available online 27th September 2004
1367-5931/$ – see front matter
# 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2004.09.001
Abbreviations
NBA L-a-amino-g-nucleobase-butyric acid
PNA peptide nucleic acid
Introduction
The nature of the molecular origins of life is a research
problem of long standing interest [1]. Major questions
include the types of polymer that may have resulted from
mixtures of the organic compounds that were present on
prebiotic earth, and how these polymers transmitted
information through cycles of growth and replication.
Current hypotheses have focused in detail, as the articles
in this issue attest, on RNA as a plausible prebiotic
progenitor. The ‘RNA world’ [2] is attractive as RNA
can be replicated directly, can store genetic information,
and has been shown to have a range of catalytic activities
[3,4]. However, there has also been great interest in
exploring other self-assembling biopolymers and nonnatural oligomers as prebiotic molecules. Excellent examples of this include hexopyranosyl analogs of RNA [5] and
peptide nucleic acids (PNA) [6].
Research into prebiotic chemistry has focused on two
main areas: the abiotic synthesis of small organic moleCurrent Opinion in Chemical Biology 2004, 8:640–644
cules that may have been the building blocks for more
complex polymers; and the design and discovery of novel
molecules and oligomers with self assembling and replicating properties. Self-assembly and replication are fundamental features of living systems and have been viewed
as necessary prerequisites for potential prebiotic molecules. Studies investigating compounds with these properties may, therefore, shed light on how living systems
evolved, and provide the basis for prebiotic hypotheses
[7].
Work initiated by Orgel [8], von Kiedrowski [9] and
others [10] has led to the realization of minimalist nucleobase systems that are capable of sustaining self-replication. Using palindromic oligonucleotides as a starting
point, these researchers designed a means by which
the product of a reaction between two smaller oligonucleotides could act as the template to promote this
reaction. Hence, the product could be formed in an
autocatalytic or self-replicating fashion. These initial
experiments have spurred interest in a wide range of
self-replicating molecules including, recently, peptides
[11,12].
The ability to control peptide oligomerization is central to
designing peptidic systems capable of sustaining selfreplication. Most work has been based upon helical,
coiled-coil peptides with well-defined pairing rules.
Coiled-coils are generally two-stranded, left-handed
superhelical motifs that have emerged as one of the
prototypical protein assembly units since their discovery
and structural elucidation 10 years ago [13]. Coiled-coils
have a striking heptad pattern (Figure 1a), with a leucine
or a suitable hydrophobic residue followed by six residues, repeated four to six times. The initial self-replicating peptide was based on the coiled-coil of the GCN4
transcription factor [11]. Whereas the oligonucleotide
self-replicating systems relied on hydrogen bonding for
precise templating, the recognition surface for template
binding in self-replicating coiled-coil peptides occurs
within the hydrophobic heptad repeat, along with electrostatic contributions from residues at the e and g positions of the heptad (Figure 1b).
The abiotic synthesis of amino acids under ‘Miller-like’
[14] experiments with less-reducing conditions [15],
and the discovery of amino acids in meteorites [16],
have provided evidence that the building blocks for
protein synthesis could have existed on primitive earth.
Experiments emulating prebiotic conditions, such as
www.sciencedirect.com
Peptide self-assembly as a model of proteins in the pre-genomic world Ghosh and Chmielewski 641
The latest in peptide self-replicators
Figure 1
(a)
Enhancing catalytic efficiency with peptide
self-replication
(b)
b
Peptide
fragments
e
a
f
Template
d
c
g
Ligation
T
B
Current Opinion in Chemical Biology
A self replication cycle based on the coiled coil motif. (a) Helical
wheel representation of a coiled-coil peptide showing the heptad
repeat. (b) The reaction cycle for a self-replicating peptide with its
fragments.
hydrothermal vents on the sea floor or bodies of water rich
in inorganic minerals with drying/wetting cycles, have
often been found to yield peptides as products [17,18].
The discovery that a peptide-based biopolymer could
promote its own synthesis opened the door to very
interesting speculations on the molecular origins of life.
With this in mind, self-replicating peptides have been
designed with many of the fundamental properties of
living systems [19,20].
In this review, we discuss the latest examples of selfreplicating peptides, with a focus on improving catalytic
efficiencies, and future directions in combining peptide
and nucleobase replicating systems. We also address
classes of newly emerging peptide-based biopolymers
with self-assembling properties, which may provide the
inspiration for self replication.
A major challenge in all replicating systems has been
attaining high catalytic efficiencies that approach exponential levels, to truly emulate natural evolution. This
difficulty is compounded by the fact that while peptide
design efforts have focused on stable assembly, the goal
of self-replication is that of achieving high catalytic
turnover as in enzyme design [21]. Two recent design
strategies, both directed towards the rational destabilization of the final product, have been utilized to overcome product inhibition (Figure 2). In an effort to
improve the catalytic efficiency of the self-replicating
peptide E1E2 [12], a three heptad repeat-containing,
coiled-coil peptide was designed, RI-26, that is one
heptad shorter than the original E1E2 sequence. The
full-length template, RI-26, was found to adopt a helical
conformation, exist as a tetramer, and have a significantly
lower Tm as compared with E1E2 (608C, versus 75%
folded at 758C). As is indicative of an autocatalytic
system, adding increasing amounts of the template
RI-26 to a reaction mixture of its fragments led to a
dramatic acceleration in product formation. A high catalytic efficiency of 1.0 105 was also observed, confirming the success of this strategy [22].
Another strategy to overcome product inhibition was to
incorporate a proline kink at the centre of a self-replicating peptide, as the fragments should maintain all interactions on either side of the kink, but the product of the
self replicating reaction should have reduced affinity for
the template. With this in mind, two proline-containing,
coiled-coil peptides were designed (XL-1 and XL-2)
based on the self-replicating peptide E1E2 [12]. Within
XL-1, a proline replaced Leu19 of E1E2, whereas in XL-2
the Glu20 residue was replaced with proline (Figure 2)
[23].
Figure 2
E1E2 Ac-ELYALEKELGALEKELACLEKELGALEKELYALEK-CONH 2
RI-26
Ac-LEKELYALEKELACLEKELYALEKEL-CONH 2
XL-1 Ac-ELYALEKELGALEKELAC PEKELGALEKELYALEK-CONH2
XL-2 Ac-ELYALEKELGALEKELACL PKELGALEKELYALEK-CONH 2
K1K2 Ac-KLYALKEKLGALKEKLACLKEKLGALKEKLYALKE-CONH 2
Ac-KLYALKE XLGALKEXLACLKEZLGALKEZLYALKE-CONH2
TA(X=TNBA, Z=ANBA), GC(X=GNBA, Z=CNBA), TT(X=TNBA, Z=ANBA)
Current Opinion in Chemical Biology
Peptide-templated RNA ligation. Sequences of self-replicating peptides (left), and a helical peptide with a proline kink (right).
www.sciencedirect.com
Current Opinion in Chemical Biology 2004, 8:640–644
642 Model systems
Both XL-1 (octamer) and XL-2 (tetramer) adopted a
helical conformation, and the helical content of their
respective peptide fragments increased upon addition of
the corresponding template. Melting temperatures of
458C and 758C were obtained for XL-1 and XL-2,
respectively, whereas E1E2 remained more than 75%
folded at 758C. These data confirm that the addition of a
proline residue to the coiled-coil at the hydrophobic d
position has a much greater effect on decreasing the
melting temperature than replacement at the hydrophilic e position. The ligation chemistry between XL-2a/b
to form XL-2 was found to proceed efficiently via selfreplication, whereas the reaction between the XL-1a/b
was very slow with less than 5% of product formed within
24 h, and added template had no effect on product
formation. The experimental data for XL-2 were analysed with the program SimFit based on the empirical
equations developed by Kiedrowski [24], and a significant catalytic efficiency of 3.2 104 was obtained [23].
This value is 260-fold greater than that obtained for
E1E2, and is comparable to the catalytic efficiency
obtained when the coiled-coil was shortened to destabilize interactions.
A peptide self-replicator containing nucelobases
To date, self-replicating peptides have been based upon
the coiled-coil motif [25] and have only utilized the
natural 20 amino acids. Recently, Mihara and co-workers
have added a new twist to peptide self-replication by
incorporating artificial L-a-amino-g-nucleobase-butyric
acids (NBAs) within the previously reported, salt-switchable self-replicating peptide K1K2 (Figure 2) [26]. The
rationale for creating this new hybrid oligomer was motivated by the success of PNA in imparting the advantages
of base-pairing alongside the stability of the amide bond.
These authors incorporated the nucleobase analogues
thymine (TNBA), adenine (ANBA), guanine (GNBA) and
cytosine (CNBA) at the complementary g–g0 positions in
the K1K2 peptide (called KK35 in this study) for possible
base-pairing in the antiparallel configuration of the
coiled-coil.
The particular peptide constructs tested incorporated two
matched sets, TA and GC, along with a control TT
(Figure 2). Each of these peptides incorporates the unnatural nucleobases in four positions in the parent and at two
positions in the two corresponding fragments that were
expected to ligate. The results of the templated reactions
showed that the relative efficiencies of the formation of
ligation products after 9 h in the presence of 10 mM added
template was in the order GC > TA > TT > KK35.
Furthermore, the authors also demonstrated that the
observed catalysis was indeed influenced by cognate
base-pairing by successfully inhibiting TA formation
by addition of exogenous adenine. It is important to note
that the auto-catalytic rate, when no template is added, is
similar for both KK35 and TA, thus pointing to significant
product inhibition.
A peptide/RNA replicating system
The ability to design complex self-replicating systems
that enable the co-existence of autocatalytic and crosscatalytic networks has been reported for both oligonucleotide and peptide systems. A further goal would be to
have a cross-catalytic system and eventually hypercyclic
system that results in the coupled replication of both a
peptide and an oligonucleotide. Such an effort would
possibly help connect the nucleic acid and peptide
worlds. Ellington and co-workers have taken a first step
in this direction by designing a system in which peptidetemplated nucleic acid ligation has been achieved in the
context of a redesigned anti-Rev RNA aptamer that is
selective for a 17-residue arginine rich motif (ARM)
found in the Rev peptide in HIV-1 (Figure 3) [27].
The aptamer was rationally dissected into two fragments
with appropriate activation chemistry to enable religation.
The ligation of the two fragments of the aptamer was
accelerated 6–10 fold in the presence of the ARM
Figure 3
(a)
(b)
Aptamer: dissection site
GA
U
5′-GGCUG CUC__GUAC U
3′-CCGAC GAG CAUG C
G
GU
AAA
(c)
Rev peptide: NH2-TRQARRNRRRRWRERQR-CO2H
Current Opinion in Chemical Biology
RNA ligation with a cationic peptide. (a) Structure of the Rev ARM peptide (ribbon in dark blue) bound to the anti-Rev aptamer (CPK in cyan)
with the site for dissection in pink [39]. (b) The sequence and secondary structure of the anti-REV aptamer with the site of dissection for future
ligation, coloured in pink. (c) The sequence of the HIV-1 Rev arginine-rich motif (residues 34–50 of HIV-1 Rev protein).
Current Opinion in Chemical Biology 2004, 8:640–644
www.sciencedirect.com
Peptide self-assembly as a model of proteins in the pre-genomic world Ghosh and Chmielewski 643
peptide. This suggests that the two RNA fragments are
preorganized upon the ARM template, enabling facile
ligation. Control ligation experiments in the presence of
other arginine-rich peptides showed no rate-enhancement, suggesting specific ternary complexation between
the ARM peptide and the two aptamer fragments. The
next step in these very interesting, linked replicating
experiments would be to appropriately dissect the
ARM peptide or designed analogues, such that ligation
is possible only in the presence of the corresponding RNA
aptamer. Thus, in such a system, neither the peptide nor
the RNA could self-replicate but the peptide–RNA system could only replicate as a whole.
Novel amide-bond-containing
self-assembling architectures
The ability to self-replicate utilizing amide-bond synthesis will certainly not be limited to canonical peptides
synthesized from a-amino acids, but will be inclusive of
most amide-bond-containing heteropolymers capable of
self-assembly. Three new heteropolymers containing
amide bonds — PNAs, peptoids and b-peptides — fulfil
the self-assembly criteria. PNAs, which mimic many of
the biophysical properties of DNA, are not discussed here
and the reader is referred to a recent review [28].
Peptoids, which are heteropolymers composed of different N-substituted glycine monomer units, were initially
designed as peptide surrogates with improved pharmacological properties [29]. More recently, Burkoth et al.
have discovered peptoids that can adopt helical secondary structures and sustain self-assembly that can probably provide novel self-replicating systems [30]. In a
different effort towards novel self-assembling systems,
Gellman and Seebach have reported the rational design,
synthesis and characterization of heteropolymers comprising b-amino acids, which contain an additional methylene between the amine and carboxyl groups of an
a-amino acid [31,32]. Recently, b-peptides that adopt
14-helix secondary structures have been redesigned to
incorporate different combinations of b-amino acid analogues of the four nucleobases [33]. One of the designed
b-peptides that incorporates the CGCG palindromic
sequence at the 2, 5, 8 and 11 positions was shown to
self-associate, presumably in an antiparallel fashion, with
a high degree of thermal stability. These nucleobasecontaining b-peptides and further redesigns of heteropolymeric b-peptides oligomers [34,35] will be amenable
for designing novel self-replicating systems. The reader is
also referred to reviews of non-peptidic heteropolymeric
systems [36,37] that can be organized into higher-order
structures and are also possible candidates for prebiotic
self-replication.
Conclusion
Self-assembling amide-based polymers and self-replicating peptides provide a testing ground for simple evoluwww.sciencedirect.com
tionary principles. The examples in this review illustrate
the major challenges and recent advances in improving
catalytic efficiency, probing new architectures, and elucidating new concepts with peptide–RNA hypercyclic
networks. Alongside these advances in experimental
work, recent theoretical work has started to interrogate
the bridge between survival-of-the-fittest and the emergence of global coexistence [38]. New experiments in
self-replicating peptides are essential for validating such
theoretical models, along with raising new and unforeseeable challenges only possible through experimentation.
Acknowledgements
Jean Chmielewski is grateful to NSF and NASA for support. Indraneel
Ghosh thanks the Donors of the Petroleum Research Fund and the
Research Corporation for support.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
1.
Brack A: The Molecular Origins of Life: Assembling the Pieces of
the Puzzle. Cambridge: Cambridge University Press; 1998.
2.
Gesteland RF, Cech TR, Atkins JF: The RNA World. New York Cold
Spring Harbor Laboratory Press; 1999.
3.
Wilson DS, Szostak JW: In vitro selection of functional nucleic
acids. Annu Rev Biochem 1999, 68:611-647.
4.
Jaschke A: Artificial ribozymes and deoxyribozymes.
Curr Opin Struct Biol 2001, 11:321-326.
5.
Eschenmoser A: Towards a chemical etiology of nucleic acid
structure. Orig Life Evol Biosph 1997, 27:535-553.
6.
Egholm M, Buchardt O, Nielsen PE, Berg RH: Peptide nucleic
acids (PNA). Oligonucleotide analogs with an achiral peptide
backbone. J Am Chem Soc 1992, 114:1895-1897.
7.
Szostak JW, Bartel DP, Luisi PL: Synthesizing life. Nature 2001,
409:387-390.
8.
Zielinski WS, Orgel LE: Autocatalytic synthesis of a
tetranucleotide analog. Nature 1987, 327:346-347.
9.
von Kiedrowski G: A self-replicating hexadeoxynucleotide.
Angew Chem Int Ed Engl 1986, 25:932-934.
10. Robertson A, Sinclair AJ, Phip D: Minimal self-replicating
systems. Chem Soc Rev 2000, 29:141-152.
11. Lee DH, Granja JR, Martinez JA, Severin K, Ghadiri MR:
A self-replicating peptide. Nature 1996, 382:525-528.
12. Yao S, Ghosh I, Zutshi R, Chmielewski J: A pH-modulated, selfreplicating peptide. J Am Chem Soc 1997, 119:10559-10560.
13. Burkhard P, Stetefeld J, Strelkov SV: Coiled coils: a highly
versatile protein folding motif. Trends Cell Biol 2001,
11:82-88.
14. Miller SL: A production of amino acids under possible primitive
earth conditions. Science 1953, 117:528-529.
15. Rode BM: Peptides and the origin of life. Peptides 1999,
20:773-786.
16. Cronin JR, Moore CB: Amino acid analysis of the Murchison,
Murray and Allende carbonaceous chondrites. Science 1971,
172:1327-1329.
17. Ogata Y, Imai E, Honda H, Hatori K, Matsuno K: Hydrothermal
circulation of seawater through hot vents and contribution
of interface chemistry to prebiotic synthesis. Orig Life Evol
Biosph 2000, 30:527-537.
Current Opinion in Chemical Biology 2004, 8:640–644
644 Model systems
18. Yokoyama S, Koyama A, Nemoto A, Honda H, Hatori K,
Matsuno K: Amplification of diverse catalytic properties of
evolving molecules in a simulated hydrothermal environment.
Orig Life Evol Biosph 2003, 33:589-595.
19. Issac R, Ham Y, Chmielewski J: The design of self replicating
helical peptides. Curr Opin Struct Biol 2001, 11:458-463.
20. Li X, Chmielewski J: Challenges in the design of self replicating
peptides. Org Biomol Chem 2003, 1:901-904.
21. Hilvert D: Critical analysis of antibody catalysis. Annu Rev
Biochem 2000, 69:751-793.
22. Issac R, Chmielewski J: Approaching exponential growth with a
self replicating peptide. J Am Chem Soc 2002, 124:6808-6809.
Truncating coiled-coil peptides destabilized the interhelical interactions
and promoted high catalytic efficiency for peptide self replication.
23. Li X, Chmielewski J: Peptide self replication enhanced by a
proline kink. J Am Chem Soc 2003, 125:11820-11821.
The use of a proline kink in a coiled-coil peptide promoted highly efficient
peptide self-replication. However, the placement of the proline kink within
the coiled coil was a critical factor in the success of this strategy. This
efficiency is competitive with that obtained for a number of enzymes. The
ease in which high catalytic efficiency can be obtained with this system
points to the possibility of obtaining self-replicating peptides with exponential growth in the future.
Peptoids - a modular approach to drug discovery.
Proc Natl Acad Sci USA 1992, 89:9367-9371.
30. Burkoth TS, Beausoleil E, Kaur S, Tang D, Cohen FE,
Zuckermann RN: Toward the synthesis of artificial proteins.
The discovery of an amphiphilic helical peptoid assembly.
Chem Biol 2002, 9:647-654.
Through combinatorial design efforts, a 15-residue amphiphilic peptoid
was obtained that self-assembled into a tetrameric structure. These
results may lead to peptoid tertiary structures in the future.
31. Appella DH, Christianson LA, Karle IL, Powell DR, Gellman SH:
beta-Peptide foldamers: robust helix formation in a new family
of beta-amino acid oligomers. J Am Chem Soc 1996,
118:13071-13072.
32. Seebach D, Overhand M, Kuhnle FNM, Martinoni B, Oberer L,
Hommel U, Widmer H: Beta-peptides: synthesis by ArndtEistert homologation with concomitant peptide coupling.
Structure determination by NMR and CD spectroscopy and
by X-ray crystallography. Helical secondary structure of a
beta-hexapeptide in solution and its stability towards pepsin.
Helv Chim Acta 1996, 79:913-941.
24. von Kiedrowski G: Minimal replicator theory I: parabolic versus
exponential growth. Bioorg Chem Front 1993, 3:113-146.
33. Bruckner AM, Chakraborty P, Gellman SH, Diederichsen U:
Molecular architecture with functionalized beta-peptide
helices. Angew Chem Intl Ed 2003, 42:4395-4399.
A beta-peptide, 14-helix is reported that acts as a scaffold for the
presentation of preorganized recognition units, such as nucleobases.
14-Helical-peptides functionalized with nucleobases form very stable
duplexes with complementary helices.
25. Takahashi Y, Mihara H: Construction of a chemically and
conformationally self replicating system of amyloid-like fibrils.
Bioorg Med Chem 2004, 12:693-699.
34. Raguse TL, Lai JR, LePlae PR, Gellman SH: Toward beta-peptide
tertiary structure: Self-association of an amphiphilic 14-helix
in aqueous solution. Org Lett 2001, 3:3963-3966.
26. Matsumura S, Takahashi T, Ueno A, Mihara H: Complementary
nucleobase interaction enhances peptide-peptide recognition
and self-replicating catalysis. Chem Eur J 2003, 9:4829-4837.
A coiled-coil peptide that included nucleobases for enhanced interpeptide interactions demonstrated self-replicative properties.
35. Cheng RP, DeGrado WF: Long-range interactions stabilize the
fold of a non-natural oligomer. J Am Chem Soc 2002,
124:11564-11565.
27. Levy M, Ellington AD: Peptide-templated nucleic acid ligation.
J Mol Evol 2003, 56:607-615.
The ligation of two fragments of an RNA aptamer is reported to be
catalyzed by a cationic Tat peptide. This work may lead to novel peptide–RNA hypercyclic networks.
28. Hyrup B, Nielsen P: Peptide nucleic acids (PNA): synthesis,
properties and potential applications. Bioorg Med Chem 1996,
4:5-23.
29. Simon RJ, Kania RS, Zuckermann RN, Huebner VD, Jewell DA,
Banville S, Ng S, Wang L, Rosenberg S, Marlowe CK et al.:
Current Opinion in Chemical Biology 2004, 8:640–644
36. Hill DJ, Mio MJ, Prince RB, Hughes TS, Moore JS: A field guide to
foldamers. Chem Rev 2001, 101:3893-4011.
37. Patch PA, Barron AE: Mimicry of bioactive peptides via
non-natural, sequence-specific peptidomimetic oligomers.
Curr Opin Chem Biol 2002, 6:872-877.
38. Stadler BMR, Stadler PF: Molecular replicator dynamics.
Adv Comp Sys 2003, 6:47-77.
39. Ye XM, Gorin A, Ellington AD, Patel DJ: Deep penetration of
an alpha helix into a widened RNA major groove in the HIV-1
rev peptide-RNA aptamer complex. Nat Struct Biol 1996,
12:1026-1033.
www.sciencedirect.com