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Project Progress

Ghadiri’s laboratory investigates the importance of Molecular Darwinistic processes in the origins of life issues by exploiting catalytic and self-reproducing (autocatalytic) polypeptide systems. The primary goal of our research program is to discover and understand factors and mechanisms that can direct the self-organization of inanimate chemical transformations into the animate chemistry of living systems. Our approach has been to rationally design and recreate various forms of autocatalytic peptide networks in the laboratory and study how the interplay of molecular information and nonlinear catalysis can lead to self-organization and expression of emergent properties. Our laboratory had reported previously that short helical peptides can efficiently self-replicate, in a template-dependant fashion, by catalyzing their own synthesis from appropriately functionalized shorter peptide fragments. Moreover, we had shown how such species can be employed to construct self-organized (auto)catalytic networks that can display some of the basic properties often associated with living systems such as selection, symbiosis, and error correction. During the first year of the NAI program, Ghadiri’s group developed An Exponential Replicator fulfilling the structural and kinetic requirements for the onset of Darwinian evolution1; established the Emergence of a Peptide Replicator in High Salt which highlights the effects of environmental factors on template-directed catalysis2; the first experimental evidence for A Chiroselective Peptide Self-Replication addressing the important issue of origin of homochirality in terrestrial proteins3; and design, synthesis, and characterization of A Reciprocal Autocatalytic Peptide Network which illustrates how self-reproduction can emerge out of mutually autocatalytic set of chemical reactions4.
In the past year Ghadiri’s NAI team has completed the chiroselective peptide replication studies supporting a fundamental hypothesis in the origin of biomolecular homochirality; designed and studied a new reciprocal self-replicating network; completed the synthetic phase of the first generation 256-member molecular ecosystem; and established the essential MALDI-TOF mass spectrometric analytical tool needed for the study of the complex networks by designing a new matrix systems for the simultaneous and quantitative analysis of multiple peptide species in complex reaction mixtures. These advances are briefly described below.
1. Chiroselective peptide self-replication. It has been argued that homochirality is a necessary condition for self-reproducing prebiotic molecular systems. However, despite its central importance in the origins of life theories, the feasibility of chiroselective amplification in biopolymers had not been established. Previous attempts aimed at demonstrating template-directed homochiral replication of nucleic acids and its analogues have proven inconclusive due to product or cross-isomer inhibition processes. We have demonstrated that a peptide replicator can amplify efficiently and specifically homochiral products through a chiroselective autocatalytic cycle. Moreover, the system is shown to be remarkably robust with even a single stereochemical mutation inhibiting the self-replication process supporting the postulate that homochirality is a necessary condition for self-replication.
(See figure 1)
Figure 1. Homochiral amplification by the two templates, TLL and TDD, through autocatalysis. TLL and TDD preorganize electrophilic and nucleophilic peptide fragments, promoting a reaction that affords another copy of TLL and TDD. This process is referred to as chiroselective because the templates only catalyze the reaction of fragments that are of the same stereochemistry as the template. The templates serve as catalysts in their own formation resulting in a nonlinear reaction rate. The nonlinear reaction rate increases the diastereomeric excess (more homochiral template than heterochiral template) over time.

2. Reciprocal self-reproducing networks. Studies on the non-enzymatic self-replication of synthetic molecules have provided a great deal of insight into the theoretical and experimental approaches to the origin of life. However, a living system is a far more complex network of chemical reactions. In attempts to further our understanding of how the complexity and emergence in living systems arise, various models of chemical networks based on self-replicating molecules have been constructed. The two most recent designs that we have completed comprise of two peptides neither capable of self-replication. However, when mixed together, the system as a whole reproduces due to the reciprocal catalysis afforded by its products. These peptides constitute the first implementations of the minimum three-component reciprocal network. It remains to be seen how such reciprocal networks behave in the context of more complex systems.


(See Figure 2)
Figure 2. Schematic representation of the self-replication cycle through reciprocal catalysis. A template T1 assembles an electrophilic fragment E and a nucleophilic fragment N2 to facilitate the ligation. The first step in the ligation involves transthiolesterification of the benzyl thioester of E by the cystein side chain to form T2* which subsequently rearranges to form a native amide bond to generate the heterodimer T1T2. Reversible dissociation of T1T2 regenerates the catalytically active monomers, completing the catalytic cycle. A similar cycle exists for T2 (cycle II).

3. Design of a 256 member molecular ecosystem. One of the major goals of our research program has been to to construct a molecular ecosystem and study the behavior of a large collection of catalytic and autocatalytic peptides, the process of self-organization, and network formation under variety of starting conditions and environmental stimuli. We have completed the synthetic phase of the first generation ecosystem. The system is composed of 16 electrophilic and 16 nucleophilic peptide fragments that when combined would yield 256 distinct mass-encoded peptide condensation products. Our immediate objective in employing this relatively large molecular population is the following: a) to investigate the production of efficient replicators, and b) to determine in how many ways and under what selection pressures complex selforganized systems may arise. Moreover, we will explore whether or not the first generation peptide ecosystem can reorganize (adapt) to changes in its environmental conditions such as pH, ionic strength, temperature, etc. The main practical obstacle in the study of a dynamic molecular ecosystem is how to find the peptides and networks of interest amongst the many non-fertile sequences and unproductive background processes that are expected to occur in such complex reaction mixtures. Current high performance chromatographic analysis techniques would not be useful in such complex cases since most of the peptides employed possess similar physical characteristics. Our strategy has been to use the rapidly advancing technology of MALDI mass spectrometry to follow the behavior of every peptide within the population and discover the peptides and network organizations of interest. By mass spectrometry one should be able to quantitatively monitor the production of each peptide if each member of the population is represented by a unique molecular mass. As described briefly below we have recently developed the required methodology to exploit mass spectrometry for the analysis of the molecular ecosystem.
4. Quantitative analysis of multicomponent catalytic and autocatalytic peptide fragment condensation reactions by MALDI-TOF mass spectrometry. The quantitative study of these multicomponent reaction systems is a challenging endeavor since most of the existing methods are either very time-consuming or limited to the analysis of a small number of species in solution. For example, reversed-phase high-performance liquid chromatography (RP-HPLC) as the method of choice for the analysis of autocatalytic networks can only be used for systems composed of approximately four starting components yielding about the same number of products. This is due to the limited resolution of HPLC and the chromatographic “similarity” of typical analytes such as DNA or peptides. As a prerequisite for a research program directed at the design, analysis, and discovery of novel self-organizing chemical networks of higher complexity, we have sought to evaluate the ability of modern matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for multicomponent reaction profile analyses. Multicomponent peptide self-replication reactions based on the templating effect of coiled-coil peptides were monitored quantitatively with MALDI-TOF-MS. A large number of matrices in combination with a variety of co-matrices were screened against a model mixture of four different peptides to obtain similar ionization intensities, despite significant differences in their amino acid composition and aggregation tendencies. The novel combination of 2â??mercaptobenzothiazole with tris(2â??carboxyethyl)phosphine as the MALDI matrix system was found to be the most efficacious in all mixtures studied. Under these conditions strong signals are observed even in the presence of large amounts of sample buffer and other contaminants. The utility of the system was demonstrated by the detailed study of several peptide replication reactions. Up to 16 individual product peptides that were formed simultaneously in a reaction mixture could be quantified accurately. The results raise the prospect of further applications of MALDI-MS for the studies of multicomponent reaction systems of even higher complexity such as the one planned for the study of molecular ecosystem as well as fast quantitative analyses in the field of proteomics and combinatorial chemistry.