PhaC-HRT2trunc

Imperial-College 2024

This part is designed by Imperial-College 2024 as a part invovled in the formation of an artifical organelle of rubber through a PHA-inspired process.

PhaC-HRT2trunc is a fusion protein combining 1. the N-terminus hydrophobic domain of poly-hydroxyalkanoate (PHA) synthase enzyme (PhaC) from C. necator; 2. the turncated HRT2 rubber synthase enzyme (HRT2trunc); and 3. a bridging peptide designed to rigidify the entire structure and positioning the HRT2trunc to the correct orientation. This enzyme is designed to produce rubber granules through a mechanism inspired by the foramtion of PHA carboxysomes inside prokaryotes.

Background

Polyhydroxyalkanoate, is a hydrophobic polymer made in many prokaryotes, and recently becoming a famous material as it can be used as a bio-degradable plastic. Whilst the polymer is very hydrophobic in nature, it can be readily produced in large quantities in prokaryotic system, comparing to rubber. Insights into the structure of the enzyme producing the PHA, the PHA synthase (PhaC), reveals the existence of a hydrophobic domain close to the active site. This domain is shown to be involved in the granule formation of the PHA and with absence of this domain, little PHA is made in the cell ^[1].

Inspired by the structure of PhaC, we questioned if the mechanism could be applied to the formation of rubber granules. Where the HRT2trunc (BBa_K5416000) is redesign with attaching the domain of PhaC1. This would hypothetically capture the newly formed rubber chain and phase-separate the rubber granule with the rest of the cell. By introducing the other PHA-associated protein, we hypothesized this structure will be then stabilized and help forming an ideal environment for rubber synthesis.

Design

Too Long Didnt Read: Here is our work flow to design this part.

Work flow of how this part is designed

This part is designed from the HRT2trunc (BBa_K5416000) by attaching the hydrophobic domian of the PhaC close to the exit of the polyisoprene chain. Whereby we begin by docking the HRT2trunc to the hydrophobic pad using online protein-protein docking serve (HADDOCK) ^[2].

Fig.1: the docked structure where the bridge peptide binds to the pad

To imporve the binding affinity of HRT2trunc to the hydrophobic pad to from a rigid structure for capturing rubber. The interface residues with opposing charges are identified and replaced with attacting charged ones ^[3].

Fig.2: close up view of the interface residues, interacting ones are colored yellow

After residues at the interface has been replaced, the structure is hence analysed for increase in stability using molecular dynamics simulation. The output structure will also be feedback and used as the template for a next round of residue-replacement. Over two cycle, we have successfully identified a significant increase in the stability of the interface, measured by decrease in RMSD of the viberating residues ^[4].

Fig 3: Two rounds of molecular dynamics simulation and residue replacement. The residues at the interface (indicated with arrows) are significantly more stablier in after replacement (deeper curve), than before (ligher curve).

Finally, the final amino acid sequence is used to predict the 3D structure of this part using Alphafold, which the predicted structure aligns with our desired configuration ^[5].

Fig 4: Alphafold 2 predicted structure after engieering

Characterization

This part is characterized in its composite format BBa_K5146030.

Protein expression

Fig.6: MOPS-Tris-Gly-SDS-PAGE profile of all BL21 transformants with different plasmids, induced overnight with 0.5mM IPTG at 25C after reaching OD =0.5. Lane L: protein precision plus dual color ladder marker Bio-rad. Lane 1: Cell lysate after induced expression of this part. Bands around 40kDa indicates the presence of PhaC-HRT2trunc

The expression of this part appears to be successful in E. coli, where a band around 40kDa is identified in lane 1, as indicated with a red arrow.

References

1. Lim H, Chuah JA, Chek MF, Tan HT, Hakoshima T, Sudesh K. Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4. International Journal of Biological Macromolecules. 2021 Sep 1;186:414-23.
2. De Vries SJ, Van Dijk M, Bonvin AM. The HADDOCK web server for data-driven biomolecular docking. Nature protocols. 2010 May;5(5):883-97.
3. Zhu J, Avakyan N, Kakkis A, Hoffnagle AM, Han K, Li Y, Zhang Z, Choi TS, Na Y, Yu CJ, Tezcan FA. Protein assembly by design. Chemical reviews. 2021 Aug 18;121(22):13701-96.
4. Kuriata A, Gierut AM, Oleniecki T, Ciemny MP, Kolinski A, Kurcinski M, Kmiecik S. CABS-flex 2.0: a web server for fast simulations of flexibility of protein structures. Nucleic acids research. 2018 Jul 2;46(W1):W338-43.
5. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nature methods. 2022 Jun;19(6):679-82.

Index

MQQRTNSLSL GQDQSDAPHP LTGAWSQLMS QTNQLLQLQS SLYQQQLGLW TQFLGQTAGN DASAPSAKPS DRRFASPEWD EHPFYSFLKQ SYLQTSKWIL ELVDKTQIDE SAKDKLSFAT RQYLRAMAPS LFMLTNPDVV KRAIETQGES LVEGMKNIVE DIQKGHDGIP THIAFILDGN GRFAKKHKLP EGGGHKAGFL ALLNVLTYCY ELGVKYATIY AFSIDNFRRK PHEVQYVMNL MLEKIEGMIM EESIINAYDI CVRFVGNLKL LDEPLKTAAD KIMRATAKNS KFVLLLAVCY TSTDEPHHHH HHPYINPYPD VLIRTSGETR LSNYLLWQTT NCILYSPHAL WPEIGLRHVV WAVQGRANDL DQNYDVNNYL LG

Above is the amino acid sequence of this part, displayed here to make future protein engineering or codon optimization easier. Domains are highlighted in different colors: PhaC (green blue #008080); HRT2trunc (deep green #003300); Intrinsic 6xHis-tag (pink #ff99cc)

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Sequence and Features

Assembly Compatibility: