Guest-protein incorporation into solvent channels of a protein host crystal (hostal)
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Guest-protein incorporation into solvent channels of a protein host crystal (hostal). / Sprenger, Janina; Carey, Jannette; Schulz, Alexander; Drouard, Fleur; Lawson, Catherine L.; Von Wachenfeldt, Claes; Linse, Sara; Leggio, Leila Lo.
In: Acta crystallographica Section D: Structural biology , Vol. 77, 2021, p. 471-485.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Guest-protein incorporation into solvent channels of a protein host crystal (hostal)
AU - Sprenger, Janina
AU - Carey, Jannette
AU - Schulz, Alexander
AU - Drouard, Fleur
AU - Lawson, Catherine L.
AU - Von Wachenfeldt, Claes
AU - Linse, Sara
AU - Leggio, Leila Lo
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - diffusion
KW - encapsulation
KW - host-guest system
KW - hostals
KW - mesopores
KW - MOLEonline
KW - protein volume fraction
KW - solvent channels
U2 - 10.1107/S2059798321001078
DO - 10.1107/S2059798321001078
M3 - Journal article
C2 - 33825708
AN - SCOPUS:85103982964
VL - 77
SP - 471
EP - 485
JO - Acta Crystallographica Section D: Structural Biology
JF - Acta Crystallographica Section D: Structural Biology
SN - 2059-7983
ER -
ID: 261148760