Linked Life Data

9893993

Source:http://linkedlifedata.com/resource/pubmed/id/9893993

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1999-2-23
pubmed:abstractText
The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the TH8/TH9 helical hairpin lies close to the bilayer surface and a second in which it inserts more deeply and appears to be transmembraneous. The loss of charge on residues E349 and D352 due to protonation at low pH has been proposed to be a critical step in transmembrane insertion, because they are within a loop connecting TH8 and TH9, and must cross the membrane upon transmembrane insertion. In this report, the role of these residues was examined by measuring the effect of the double substitution E349K/D352K on the conformation of the TH8/TH9 hairpin through a fluorescent group attached to TH9. At pH 4.5, there was shallower insertion of TH8/TH9 of the E349K/D352K mutant relative to T domain with wild-type residues at 349 and 352. In addition, smaller and/or fewer pores were obtained with the E349K/D352K mutant relative to the wild-type. On the other hand, high T domain concentrations, or further decreasing pH, allowed transmembrane insertion of both the wild-type and the 349K/352K mutant as well as induction of larger and/or more numerous pores. Furthermore, the transmembrane insertion process was rapid for both the mutant and wild-type. This shows that the mutant has the capacity to form a transmembrane structure similar to that of the wild-type T domain and, thus, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane movement. The linkage between the ability of the T domain to form the transmembrane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability to insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues other than E349 and D352 whose protonation promotes transmembrane insertion.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
http://linkedlifedata.com/resource/pubmed/chemical/1-stearoyl-2-linoleoylphosphatidylch..., http://linkedlifedata.com/resource/pubmed/chemical/4,4-difluoro-4-bora-3a,4a-diaza-s-in..., http://linkedlifedata.com/resource/pubmed/chemical/Bicyclo Compounds, Heterocyclic, http://linkedlifedata.com/resource/pubmed/chemical/Boron Compounds, http://linkedlifedata.com/resource/pubmed/chemical/Cyclic N-Oxides, http://linkedlifedata.com/resource/pubmed/chemical/Diphtheria Toxin, http://linkedlifedata.com/resource/pubmed/chemical/Fluorescent Dyes, http://linkedlifedata.com/resource/pubmed/chemical/Lipid Bilayers, http://linkedlifedata.com/resource/pubmed/chemical/Lysine, http://linkedlifedata.com/resource/pubmed/chemical/Membrane Proteins, http://linkedlifedata.com/resource/pubmed/chemical/Phosphatidylcholines, http://linkedlifedata.com/resource/pubmed/chemical/Spin Labels, http://linkedlifedata.com/resource/pubmed/chemical/TEMPO, http://linkedlifedata.com/resource/pubmed/chemical/bimanes
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
976-84
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:9893993-Bicyclo Compounds, Heterocyclic, pubmed-meshheading:9893993-Biological Transport, pubmed-meshheading:9893993-Boron Compounds, pubmed-meshheading:9893993-Cyclic N-Oxides, pubmed-meshheading:9893993-Diphtheria Toxin, pubmed-meshheading:9893993-Fluorescence Polarization, pubmed-meshheading:9893993-Fluorescent Dyes, pubmed-meshheading:9893993-Hydrogen-Ion Concentration, pubmed-meshheading:9893993-Lipid Bilayers, pubmed-meshheading:9893993-Lysine, pubmed-meshheading:9893993-Membrane Proteins, pubmed-meshheading:9893993-Mutagenesis, Site-Directed, pubmed-meshheading:9893993-Phosphatidylcholines, pubmed-meshheading:9893993-Protein Conformation, pubmed-meshheading:9893993-Protein Structure, Secondary, pubmed-meshheading:9893993-Spectrometry, Fluorescence, pubmed-meshheading:9893993-Spin Labels
pubmed:year
1999
pubmed:articleTitle
Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin.
pubmed:affiliation
Department of Biochemistry and Cell Biology, S.U.N.Y. at Stony Brook 11794-5215, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.