Linked Life Data

11900531

Source:http://linkedlifedata.com/resource/pubmed/id/11900531

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
2002-3-19
pubmed:abstractText
A fusion protein in which the C-terminus of Halobacterium salinarum sensory rhodopsin I (SRI) is connected by a flexible linker to the N-terminus of its transducer (HtrI) was constructed and expressed in H. salinarum. The fusion protein mediated attractant responses to orange light and repellent responses to UV/violet light that were comparable to those produced by the wild-type SRI-HtrI complex. Immunoblot analysis of H. salinarum membrane proteins demonstrated intact fusion protein and no detectable proteolytic cleavage products. Rapid oxidative cross-linking of a monocysteine mutant in the HtrI domain confirmed that the fusion protein exists as a homodimer in the membrane. HtrI-free SRI and HtrI-complexed SRI have been shown previously to exhibit large differences in the pH dependence of their photocycle kinetics and in the pK(a) of Asp76 that controls a pH-dependent spectral transition in SRI. These differences were used to assess whether only one or both SRI domains in the fusion protein were complexed properly to the HtrI homodimer. Measurement of the photochemical activity, the photocycle kinetics, and the absorption spectra at various pH values established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. Closer examination of the HtrI effect on SRI revealed that Asp76 titration in HtrI-free SRI fits two pK(a) values, with 98% and 2% of the molecules titrating with pK(a)'s of 7 and 9, respectively. The same two pK(a)'s of Asp76 are evident in HtrI-complexed SRI, but with 13% with pK(a) of 7 and 87% with pK(a) of 9 and a similar bias toward the pK(a) of 9 in the fusion protein. Titration of the fusion protein with Ala substitution at Arg73, a residue in the photoactive site, in the SRI domain indicates that a basic residue at Arg73 is necessary for the lower pK(a) to be observed. A model in which Arg73 plays a role in the HtrI effect on SRI is discussed.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
26
pubmed:volume
41
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3891-6
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2002
pubmed:articleTitle
Demonstration of 2:2 stoichiometry in the functional SRI-HtrI signaling complex in Halobacterium membranes by gene fusion analysis.
pubmed:affiliation
Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, 6431 Fannin Street, Houston, Texas 77030, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.