Pore structure controls stability and molecular flux in engineered protein cages

Adamson, L.; Tasneem, N.; Andreas, M. P.; Close, W.; Szyszka, T. N.; Jenner, E.; Young, R.; Cheah, L. C.; Norman, A.; Sainsbury, F.; Giessen, T. W.; Lau, Y. H.

doi:10.1101/2021.01.27.428512

bioRxiv · 10.1101/2021.01.27.428512

Pore structure controls stability and molecular flux in engineered protein cages

Adamson, L.·Tasneem, N.·Andreas, M. P.·Close, W.·Szyszka, T. N.·Jenner, E.·Young, R.·Cheah, L. C.·Norman, A.·Sainsbury, F.·Giessen, T. W.·Lau, Y. H.

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Abstract

Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. While pores lined with negatively charged residues resulted in more robust assemblies than their corresponding positively charged variants, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures of pore variants elucidated at resolutions between 2.5-3.6 [A]. Alongside these structures, molecular dynamics simulations and stopped flow kinetics experiments reveal the importance of considering both pore size and surface charge, together with flexibility and rate determining steps, when designing protein cages for controlling molecular flux. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/428512v3_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@725e76org.highwire.dtl.DTLVardef@97a497org.highwire.dtl.DTLVardef@1b6ea1forg.highwire.dtl.DTLVardef@1c4d17f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Adamson, L., Tasneem, N., Andreas, M. P., Close, W., Szyszka, T. N., Jenner, E., Young, R., Cheah, L. C., Norman, A., Sainsbury, F., Giessen, T. W., Lau, Y. H.. 2021-01-27. Pore structure controls stability and molecular flux in engineered protein cages. https://doi.org/10.1101/2021.01.27.428512

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