Accessibility of introduced cysteines in chemoreceptor transmembrane helices reveals boundaries interior to bracketing charged residues

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 ␣-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 cysteineaccessibility 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 Much is known about the structure and structural changes of the transmembrane chemoreceptors from Escherichia coli (Falke and Hazelbauer 2001). However, as is the case for many transmembrane proteins, we do not know the precise boundaries of the protein segments embedded in the hydrophobic environment of the lipid bilayer. Chemoreceptors are transmembrane homodimers that are constructed of extended helical bundles (Fig. 1A). Each monomer in the homodimer has two transmembrane segments, TM1 and TM2, which traverse the cytoplasmic membrane as helices. These two regions contain sequences of exclusively hydrophobic residues longer than the ∼20 necessary for a helix to span a biological membrane. For instance, the TM1 and TM2 regions of chemoreceptor Trg are defined by sequences of 30 and 24 hydrophobic residues, respectively.Transmembrane ␣-helices are readily predicted in protein sequences by identification of stretches of exclusively or predominantly hydrophobic residues a minimum of ∼20 residues in length (Engelman et al. 1986; von Heijne 1992;Jones et al. 1994;Rost et al. 1995). The predictions are based on the 1.5 Å distance between residues in a canonical ␣-helix and the ∼30 Å width of the hydrophobic part of a membrane (Linden et al. 1977;Seelig and Seelig 1980;Wiener and White 1992;White and Wiener 1996). HowReprint requests to: Gerald L. Hazelb...

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