Difference between revisions of "Part:BBa K2912000"

Revision as of 14:09, 11 October 2022

RebA may act as a scaffolding protein to facilitate the major polymerization process

2019 SZU-China

Biology

SZU-China 2019 iGEM team was going to find a suicide switch inside the E coli that can break the whole body of the bacteria leading to the release of RNAi molecules transcribed from E coli inducing by IPTG or some others. Therefore, we were in need of a useful mechanism. Fortunately, we finally found the Refractile inclusion bodies (R-bodies) to kill the E coli, causing the inclusion to flow out of the plasma membrane so that we can get the RNAi molecules transcribed by E coli.

Refractile inclusion bodies, known as R bodies, are produced by only a few species of bacteria. These inclusion bodies are highly insoluble protein ribbons, typically seen coiled into cylindrical structures within the cell[1]. R-bodies are produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts a phenomenon called the killer trait[2]. This is one of the DNA sequences for the R body locus (reb) from Caedibacter taeniospiralis. It has been suggested that Reb A may act as a scaffolding protein to facilitate the major polymerization process. The identity in amino acid sequence between Reb A and Reb B suggests a similar structure and function. Like Reb B, Reb A is modified into two or more species with different molecular weights before the major polymerization event occurs[3].

Comparison of the hydropathy plots for Reb B with those for Reb A suggests a similar secondary structure for these regions. Therefore, Reb A should be capable of entering either a temporary or permanent association with the polymerized complexes of Reb B. If the acidic Reb A proteins are linked to the growing R body complex (as scaffolding or at the site of polymerization), they may be responsible for the increased pIs during the major polymerization event. That is, the proportional contribution of Reb A may decline as polymerization proceeds, resulting in a shift of the pls. Pulse-chase analysis of the protein products encoded by pBQ65 reveals that, like Reb B, the modified faster-migrating species of Reb A decrease in concentration over time as the higher-molecular-weight polymerization complexes are formed. This is evidence that Reb A proteins may associate directly with the polymerization complexes[3].

The R bodies of C. taeniospiralis are type 51. They are about 0.5 μm wide, have a maximum length of 20 μm, and 13 nm thick, possess acute angles at each end and unroll in a telescopic fashion when exposed to a pH of 6.5 or lower. These proteinaceous ribbons are rolling up inside the cell to form a hollow cylinder about 0.5 μm in diameter and 0.5 μm long[4]. For more information, please see BBa_K2912017-R-body.

Sequence

Sequence and Features

2022 SZU-China

The 2022 SZU-China team conducted more in-depth research on the nature of the protein. We used a variety of bioinformatics tools to help us understand this protein. We hope that this survey will provide a wealth of information for other teams who need to use R-body to help them use it.

Basic physical and chemical properties

Number of amino acids: 115

Molecular weight: 11754.16

Theoretical pI: 3.62 Total number of negatively charged residues (Asp + Glu): 9 Total number of positively charged residues (Arg + Lys): 2 Extinction coefficients:

This protein does not contain any Trp residues. Experience shows that this could result in more than 10% error in the computed extinction coefficient.

Extinction coefficients are in units of M(-1) cm(-1), at 280 nm measured in water.

Ext. coefficient 1490 Abs 0.1% (=1 g/l) 0.127 Instability index:

The instability index (II) is computed to be 7.98 This classifies the protein as stable.

Aliphatic index: 80.70

Grand average of hydropathicity (GRAVY): 0.079

3D structure

According to AlphaFold structure predictions, the protein structure below has been predicted by DeepMind with AlphaFold (Jumper, J et al. 2021). For more information and additional features, please visit this sequence's page at AlphaFold DB.

Interaction

RebA can interact with RebB, RebC, and a variety of modifier proteins.

number of nodes: 11

number of edges: 40

average node degree: 7.27

avg. local clustering coefficient: 0.89

expected number of edges: 10

PPI enrichment p-value: 2.46e-12

Reference

[1]Koehler L, Flemming FE, Schrallhammer M. Towards an ecological understanding of the killer trait - A reproducible protocol for testing its impact on freshwater ciliates. Eur J Protistol. 2019 Apr;68:108-120. doi: 10.1016/j.ejop.2019.02.002. Epub 2019 Feb 12. PMID: 30826731.

[1]Wang B, Lin YC, Vasquez-Rifo A, Jo J, Price-Whelan A, McDonald ST, Brown LM, Sieben C, Dietrich LEP. Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence. Nat Commun. 2021 Jul 29;12(1):4613. doi: 10.1038/s41467-021-24796-0. PMID: 34326342; PMCID: PMC8322103.

[3]Heruth DP, Pond FR, Dilts JA, Quackenbush RL. Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116. J Bacteriol. 1994 Jun;176(12):3559-67. doi: 10.1128/jb.176.12.3559-3567.1994. PMID: 8206833; PMCID: PMC205544.

[4]Pond FR, Gibson I, Lalucat J, Quackenbush RL. R-body-producing bacteria.Microbiol Rev. 1989 Mar;53(1):25-67. doi: 10.1128/mr.53.1.25-67.1989. PMID:2651865; PMCID: PMC372716.

[5] Winter MA, Guhr KN, Berg GM. Impact of various body weights and serumcreatinine concentrations on the bias and accuracy of the Cockcroft-Gaultequation. Pharmacotherapy. 2012 Jul;32(7):604-12. doi:10.1002/j.1875-9114.2012.01098.x. Epub 2012 May 10. PMID: 22576791.

[6]Matsuoka JI, Ishizuna F, Kurumisawa K, Morohashi K, Ogawa T, Hidaka M, SaitoK, Ezawa T, Aono T. Stringent Expression Control of Pathogenic R-body Productionin Legume Symbiont Azorhizobium caulinodans. mBio. 2017 Jul25;8(4):e0071517. doi: 10.1128/mBio.00715-17. PMID: 28743814; PMCID:PMC5527310.

[7]White DW, Tartaglia LA. Leptin and OB-R: body weight regulation by a cytokinereceptor. Cytokine Growth Factor Rev. 1996 Dec;7(4):303-9. doi:10.1016/s1359-6101(96)00040-8. PMID: 9023054.

[8]Matsuoka JI, Ishizuna F, Ogawa T, Hidaka M, Siarot L, Aono T. Localization ofthe reb operon expression is inconsistent with that of the R-body production inthe stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR. J Gen Appl Microbiol. 2019 Sep 14;65(4):209-213. doi:10.2323/jgam.2018.09.003. Epub 2019 Feb 5. PMID: 30726794.