The functionally active Mistic-fused histidine kinase receptor, EnvZ.
Mistic is a small Bacillus subtilis protein which is of current interest to the field of structural biology and biochemistry because of its unique ability to increase integral membrane protein yields in Escherichia coli expression. Using the osmosensing histidine kinase receptor, EnvZ, an E. coli two-component system, and its cytoplasmic cognate response regulator, OmpR, we provide the first evidence that a Mistic-fused integral membrane protein maintains functionality both in vitro and in vivo. When the purified and detergent-solubilized receptor EnvZ is fused to Mistic, it maintains the ability to autophosphorylate on residue His(243) and phosphotransfers to residue Asp(55) located on OmpR. Functionality was also observed in vivo by means of a β-galactosidase assay in which RU1012 [Φ(ompC-lacZ)10-15, ΔenvZ::Km(r)] cells transformed with Mistic-fused EnvZ led to an increase in downstream signal transduction events detected by the activation of ompC gene expression. These findings illustrate that Mistic preserves the functionality of the Mistic-fused cargo protein and thus provides a beneficial alternate approach to study integral membrane proteins not only by improving expression levels but also for direct use in functional characterization.
Blain
Katherine Y
KY
Structural Biology Laboratory, The Salk Institute, La Jolla, California 92037, United States.
Kwiatkowski
Witek
W
Choe
Senyon
S
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C113724:OmpC protein; C109237:OmpF protein; D018272:Porins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C057843:envZ protein, E coli