Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain.
The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.
Kachel
K
K
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
Ren
J
J
Collier
R J
RJ
London
E
E
D001896:Boron Compounds / Q000276:immunology; D001643:Bridged Bicyclo Compounds; D004167:Diphtheria Toxin / Q000737:chemistry*; D005456:Fluorescent Dyes; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry*; D010713:Phosphatidylcholines; D017433:Protein Structure, Secondary
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D001643:Bridged Bicyclo Compounds; D004167:Diphtheria Toxin; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010446:Peptide Fragments; D010713:Phosphatidylcholines; C017251:1,2-oleoylphosphatidylcholine