In-Cell Association of a Bioorthogonal Tubulin

Yuhan Wang, Mahima Unnikrishnan, Brooke Ramsey, Driss El Andlosy, Alex T. Keeley, Catherine J. Murphy, Martin Gruebele

Research output: Contribution to journalArticlepeer-review


Studies of proteins from one organism in another organism’s cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro.

Original languageEnglish (US)
Pages (from-to)1282-1290
Number of pages9
Issue number2
StatePublished - Feb 12 2024

ASJC Scopus subject areas

  • Bioengineering
  • Materials Chemistry
  • Polymers and Plastics
  • Biomaterials


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