Team:Kent/Composite Part

Composite Parts

Envirowire: Sup35NM with an N-terminal CsgAss signal sequence and a C-terminal Cytochrome b₅₆₂
Part name: BBa_K1739003

We have made a composite part which is a fusion protein of two of our BioBricks, BBa_K1739002 (CsgA_ss-Sup35NM) and BBa_K1739001 (Cytochrome b₅₆₂).

(Figure 1. Illustrates the construction of BBa_K1739003. This image was created using SnapGene Viewer.)

This BioBrick encodes for the Envirowire fusion protein. It contains three segments, CsgA signal sequence, Sup35NM ( BBa_K1739000 and BBa_K1739002) and Cytochrome b₅₆₂ (BBa_K1739001) The bipartite CsgA signal sequence targets the Sec protein export pathway followed by the endogenous curli export system of E.coli allowing our protein to be easily exported into an external medium ^[6][7]. Sup35NM is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid ^[3][4]. This has previously been discussed by Tessier and Lindquist (2009)^[8] who show that two beta-sheets bond together in a self-complimenting ‘steric zipper’ that excludes water, leaving a highly stable parallel beta-sheet with one molecule every 4.7Å. The particular advantage of using Sup35-NM is that in its native state Sup35p has two functional domains, the N and C terminal, separated by the highly charged M domain ^[3][4][9]. Here, with the removal of the functional C domain allows us to add our own functional protein, in this case cytochrome b₅₆₂, to form a fusion protein. We chose Cytochrome b₅₆₂ as the electron carrier to make our amyloid conductive. The structure of cytochrome b₅₆₂ consists of a single 24kDa subunit containing four nearly parallel alpha helices ^[1][5]. B-type cytochromes are a favourable choice because heme binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 ^[9]. Heme binding has been shown to occur in both the native protein and the denatured protein, although the latter exhibits a modest affinity with a dissociation constant (Kd) of 3μM. This allows the cytochrome to be exported in an unfolded state and heme to be added exogenously to initiate correct folding of the cytochrome by burying hydrophobic side chains ^[2]. Furthermore, heme binding to cytochrome b₅₆₂ has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C ^[2]. This BioBrick part has been inserted into the pSB1C3 backbone and uses the promoter BBa_J23104. This part can be used in the VS45 strain of E.coli containing deletions that prevent amyloid from binding to the outside of the cell and increase the rate of protein exiting the cell via the curli export system.

References

[1] Fujiwara, T., Fnkumori,, Y. and Yamanaka, T. (1993). Halobacterium halobium Cytochrome b-558 and Cytochrome b-562: Purification and Some Properties. J. Biochem., 113, pp.48-54.

[2]Robinson, C., Liu, Y., Thomson, J., Sturtevant, J. and Sligar, S. (1997). Energetics of Heme Binding to Native and Denatured States of Cytochrome b 562 †. Biochemistry, 36(51), pp.16141-16146.

[3] Frederick, K., Debelouchina, G., Kayatekin, C., Dorminy, T., Jacavone, A., Griffin, R. and Lindquist, S. (2014). Distinct Prion Strains Are Defined by Amyloid Core Structure and Chaperone Binding Site Dynamics. Chemistry & Biology, 21(2), pp.295-305.

[4] Glover, J., Kowal, A., Schirmer, E., Patino, M., Liu, J. and Lindquist, S. (1997). Self-Seeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae. Cell, 89(5), pp.811-819.

[5] Mathews, F. S., Bethge, P. H., & Czerwinski, E. W. (1979). The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution. Journal of Biological Chemistry, 254(5), 1699-1706.

[6] Sivanathan, V. and Hochschild, A. (2012). Generating extracellular amyloid aggregates using E. coli cells. Genes & Development, 26(23), pp.2659-2667

[7] Sivanathan, V. and Hochschild, A. (2013). A bacterial export system for generating extracellular amyloid aggregates. Nat Protoc, 8(7), pp.1381-1390.

[8]Tessier, P. and Lindquist, S. (2009). Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+]. Nat Struct Mol Biol, 16(6), pp.598-605.

[9] Wickner, R., Edskes, H., Shewmaker, F. and Nakayashiki, T. (2007). Prions of fungi: inherited structures and biological roles. Nature Reviews Microbiology, 5(8), pp.611-618.