Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach.
The twin-arginine-translocase (Tat) can transport proteins in their folded state across bacterial or thylakoid membranes. In Bacillus subtilis the Tat-machinery consists of only two integral (inner) membrane proteins, TatA and TatC. Multiple copies of TatA are supposed to form the transmembrane channel, but little structural data is available on this 70-residue component. We used a multi-construct approach for expressing several characteristic fragments of TatA(d), to determine their individual structures and to cross-validate them comprehensively within the architecture of the full-length protein. Here, we report the design, high-yield expression, detergent-aided purification and lipid-reconstitution of five constructs of TatA(d), overcoming difficulties associated with the very different hydrophobicities and sizes of these membrane protein fragments. Circular dichroism (CD) and oriented CD (OCD) were used to determine their respective conformations and alignments in suitable, negatively charged phospholipid bilayers. CD spectroscopy showed an N-terminal alpha-helix, a central helical stretch, and an unstructured C-terminus, thus proving the existence of these secondary structures in TatA(d) for the first time. The OCD spectra demonstrated a transmembrane orientation of the N-terminal alpha-helix and a surface alignment of the central amphiphilic helix in lipid bilayers, thus supporting the postulated topology model and function of TatA as a transmembrane channel.
Forschungszentrum Karlsruhe, POB 3640, D-76021 Karlsruhe, Germany.
Christian
C
Lange
Sonja D
SD
Müller
Torsten H
TH
Walther
Jochen
J
Bürck
Anne S
AS
Ulrich
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C499697:TatA protein, E coli