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Peptide based self-replicating coacervate protocells

Periodic Reporting for period 1 - PEPREP (Peptide based self-replicating coacervate protocells)

Reporting period: 2019-07-16 to 2021-07-15

The main challenge in designing the coacervates-based protocells in the chemistry laboratory is the complexity in the constituting building blocks of design protocells. Usually, these complex coacervates are formed from the combination of two components, which are bigger in molecular weight and give rise to the complex understanding of their assembly process being used for the protocell studies. Recently, it has been observed that many research groups are developing minimal biomolecules for the formation of complex coacervates, which could be used as a protocell model. They are mainly focussing on the simple and short motifs including nucleotide bases, short peptides, and metals to make the complex coacervates with traditional electrostatic interaction. There are main three problems in the complex coacervates systems: 1) structural complexity, 2) longer and bigger molecules rise the difficulties to understand the formation process, 3) they are mostly not prebiotic relevant.
By keeping the challenges in mind, our ultimate goal was to design and synthesize the simplest single molecule-based coacervates to make the protocells in the laboratory. Our design is made of short peptide derivatives and can be synthesized accurately with great reproducibility in four steps in an ordinary chemistry laboratory. We prepared several derivatives with a sticker-spacer model approach, where we use the hydrophobic amino acids as stickers and different spacers as linkers between two stickers. The model compound (FFssFF) was formed with diphenylalanine (FF)-spacer as the apolar motif and disulfide linker (cystamine) as a spacer as the polar motif. The model compound is soluble in water and on increasing the pH it forms the coacervates and looks like a turbid solution in the vial and was confirmed under the microscope.
The project has set the foundational work for an entirely new direction and these results will benefit society with a great impact in the future.
The main objectives of this project were divided into four steps, which correspond to respective work packages (WPs). The following are the brief objectives of the project.
1- Design and synthesis of the peptide derivatives
2- Coacervation, Different linkers with different physical and chemical properties, including the different chemistries of linkers for replication
3- Encapsulation of macromolecules into coacervates
4- Coacervates as microreactors and coacervates to vesicle transition
Experimental work
I briefly described the work done in the duration of the project.

Work Package 1: Design and synthesis of short peptide building blocks. The design, synthesis, and analysis of our model peptide derivative are summarised in deliverables, which are included separately. Moreover, we have shown that our design can easily be extended to several different combinations of amino acids.
We have synthesized several different peptides conjugates that can be used to form coacervates,
- FFssFF molecules were used as a model compound
- We exchanged the phenylalanine with other hydrophobic amino acids with increasing and decreasing the hydrophobic ability of the sticker residues.
- The spacers/linkers between two hydrophobic stickers residues were also changes in several molecules to see the effect of polarity of spacers on coacervation.

Work package 2: Coacervation and characterization
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The model compound FFssFF and most of the analogue derivatives are water-soluble compounds. We made the 1 to 15 mg/ml concentration as stock solutions in Milli-Q and increased the pH by using NaOH or any buffer of pH 7.0. This immediately converted the transparent solution to turbid solution as shown in fig 4 and turbidity is responsive towards pH. Under the microscope, we observed the coacervates in turbid solution while there were no coacervates in the transparent solution at pH-6.

Work package 3: Reversibility and Encapsulation of the pigments
In most of the compounds, we focus on the spacer of cystamine, which is a hydrophilic linker. The interesting point of this linker is not only the hydrophilicity to drive the liquid-liquid phase separation but also its redox responsive nature plays a role to make the system more prebiotically relevant. We used the reducing agents DTT and TCEP to reduce the disulfide bond which ultimately dissolve the coacervates and turbidity also gone on reduction.

Work package 4: Creation of functional protocells

Coacervates of diphenylalanine dipeptides (FF-ss-FF) have been found to take up a wide range of prebiotically interesting molecules, like short RNAs, ssDNA, and natural pigments. In this WP-4,
In the report, I investigated the promising results of coacervates to vesicles transformation which deviates us to go in this direction before exploring the replication property of the coacervates. One of the analogue derivatives of FFssFF, where we replaced the phenylalanine with tyrosine and tyrosine is well-known amino acid for enzymatic oxidation. The YFsFY compound form the coacervates in a similar way as FFssFF does form but interestingly by adding the oxidizing agents, the coacervates transform into the semipermeable membrane and we could say the internal core of vesicles/shells is still mobile and similar to the coacervates.
For all oral and poster presentations, cluster meetings, and research papers, we acknowledged the Marie Curie Foundation for the great financial support.
Progress beyond the state of the art and potentials impacts

1. Impact

The information on Impact in section 2 of the proposal is still relevant and does not require updating. The impact this project has had so far on the fellow, the host institute, and the European research agenda can be summarized as follows.
With very unfortunate restrictions on travel and physical conferences, it was difficult to interact with renowned scientists and experts in the fields. However, thanks to the world of IT, I manage to have interactions with different groups from UK, Netherlands, and China to build an extensive scientific network.

2. Exploitation and dissemination of results

The plan for exploitation and dissemination of results as described in the project does not require an update. So far, our results have been disseminated within the scientific community on various forums and through social media (Twitter, Linked In, Facebook) platforms.
As I took one writing course in the language department which helped me a lot to improve my writing. Then I planned to write a review article on the relevant field which was published in chemical society review see reference 2. This article has improved my skills, knowledge about the subject and somehow made me an expert in the field.
- The poster was presented at the physical conference in 2019 on CHIAN (Eindhoven).
- An oral presentation was given at the conference on CHAIN in 2020 (Eindhoven).
- In two invited talks, the results were presented to graduate students, and one talk was streamed online on Facebook for the public.
In addition, we are preparing a paper for publication in a peer-reviewed journal about the in-situ formation of membranes outside the coacervates.
Chemical structure of model compound and microscopic image