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Published online before print May 7, 2004
Protein Science, DOI: 10.1110/ps.04648604
 Copyright © 2004 The Protein Society
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Accessibility of introduced cysteines in chemoreceptor transmembrane helices reveals boundaries interior to bracketing charged residues

Thomas Boldog and Gerald L. Hazelbauer

Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri 65211, USA

(RECEIVED January 28, 2004; FINAL REVISION February 18, 2004; ACCEPTED February 18, 2004)



Abstract

Two hydrophobic sequences, 24 and 30 residues long, identify the membrane-spanning segments of chemoreceptor Trg from Escherichia coli. As in other related chemoreceptors, these helical sequences are longer than the minimum necessary for an {alpha}-helix to span the hydrocarbon region of a biological membrane. Thus, the specific positioning of the segments relative to the hydrophobic part of the membrane cannot be deduced from sequence alone. With the aim of defining the positioning for Trg experimentally, we determined accessibility of a hydrophilic sulfhydryl reagent to cysteines introduced at each position within and immediately outside the two hydrophobic sequences. For both sequences, there was a specific region of uniformly low accessibility, bracketed by regions of substantial accessibility. The two low-accessibility regions were each 19 residues long and were in register in the three-dimensional organization of the transmembrane domain deduced from independent data. None of the four hydrophobic–hydrophilic boundaries for these two membrane-embedded sequences occurred at a charged residue. Instead, they were displaced one to seven residues internal to the charged side chains bracketing the extended hydrophobic sequences. Many hydrophobic sequences, known or predicted to be membrane-spanning, are longer than the minimum necessary helical length, but precise membrane boundaries are known for very few. The cysteine-accessibility approach provides an experimental strategy for determining those boundaries that could be widely applicable.

Keywords: bacterial chemoreceptors; transmembrane proteins; cysteine scanning; lipid bilayers; hydrophobic–hydrophilic boundaries

Reprint requests to: Gerald L. Hazelbauer, Department of Biochemistry, University of Missouri–Columbia, 117 Schweitzer Hall, Columbia, MO 65211, USA; e-mail: hazelbauerg{at}missouri.edu; fax: (573) 882-5635.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04648604.


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