Relative domain folding and stability of a membrane transport protein.

Journal of molecular biology (2014-02-18)
Nicola J Harris, Heather E Findlay, John Simms, Xia Liu, Paula J Booth

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately +2 kcal mol(-1). There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of +2 kcal mol(-1). This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.

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L-Tryptophan, reagent grade, ≥98% (HPLC)
L-Tryptophan, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 99.0-101.0%
L-Tryptophan, BioUltra, ≥99.5% (NT)
Tryptophan, Pharmaceutical Secondary Standard; Certified Reference Material
L-Tryptophan, certified reference material, TraceCERT®
Tryptophan, European Pharmacopoeia (EP) Reference Standard