Guest-protein incorporation into solvent channels of a protein host crystal (hostal)

Janina Sprenger, Jannette Carey, Alexander Schulz, Fleur Drouard, Catherine L. Lawson, Claes Von Wachenfeldt, Sara Linse, Leila Lo Leggio

Research output: Contribution to journalArticlepeer-review

Abstract

Soaking small molecules into the solvent channels of protein crystals is the most common method of obtaining crystalline complexes with ligands such as substrates or inhibitors. The solvent channels of some protein crystals are large enough to allow the incorporation of macromolecules, but soaking of protein guests into protein crystals has not been reported. Such protein host crystals (here given the name hostals) incorporating guest proteins may be useful for a wide range of applications in biotechnology, for example as cargo systems or for diffraction studies analogous to the crystal sponge method. The present study takes advantage of crystals of the Escherichia coli tryptophan repressor protein (ds-TrpR) that are extensively domain-swapped and suitable for incorporating guest proteins by diffusion, as they are robust and have large solvent channels. Confocal fluorescence microscopy is used to follow the migration of cytochrome c and fluorophore-labeled calmodulin into the solvent channels of ds-TrpR crystals. The guest proteins become uniformly distributed in the crystal within weeks and enriched within the solvent channels. X-ray diffraction studies on host crystals with high concentrations of incorporated guests demonstrate that diffraction limits of ∼2.5 Å can still be achieved. Weak electron density is observed in the solvent channels, but the guest-protein structures could not be determined by conventional crystallographic methods. Additional approaches that increase the ordering of guests in the host crystal are discussed that may support protein structure determination using the hostal system in the future. This host system may also be useful for biotechnological applications where crystallographic order of the guest is not required.

Original languageEnglish (US)
Pages (from-to)471-485
Number of pages15
JournalActa Crystallographica Section D: Structural Biology
Volume77
DOIs
StatePublished - Apr 1 2021

All Science Journal Classification (ASJC) codes

  • Structural Biology

Keywords

  • MOLEonline
  • diffusion
  • encapsulation
  • host-guest system
  • hostals
  • mesopores
  • protein volume fraction
  • solvent channels

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