Stable folding core in the folding transition state of an α-helical integral membrane protein

Curnow, Paul; Bartolo, Natalie Di; Moreton, Kathleen M.; Ajoje, Oluseye O.; Saggese, Nicholas P.; Booth, Paula J.

doi:10.1073/pnas.1012594108

Cited by 48 publications

(50 citation statements)

References 49 publications

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“…Insertion of the second, C domain of LacY does not appear to require the equivalent regions of the domain at the initial helix of the C domain to have enhanced stability. This is despite the fact 13 that that the C domain can be expressed and folded in the absence of the N domain 32 , and that the two domains exhibit pseudo symmetry and are thought to arise from gene duplication 33 .…”

Section: Relative Stability Of Helix I and Viimentioning

confidence: 99%

Relative Domain Folding and Stability of a Membrane Transport Protein

Harris

Findlay

Simms

et al. 2014

Journal of Molecular Biology

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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 ~ +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. 1 Relative domain folding and stability of a membrane transport proteinHarris et al. Response to referees' commentsWe thank the referees for their helpful comments and have made changes to the manuscript in accordance with the issues raised by the editor and referees as detailed below. Additionally to improve clarity we have made colour version of the figures. EditorWe have re-written the abstract and expanded the explanation of the single Trp mutants, p2&5.It is hard to comment on the absolute magnitude of the free energy value determined for LacY and GalP, in the absence of thermodynamic measurements on other membrane proteins as well as studies using different methods to assess thermodynamic stability in lipid bilayers and kinetic stability. The free energy is small implying that the inherent protein structure has relatively low thermodynamic stability. It could be that the bilayer increases the kinetic stability, or that the absolute magnitude is partly dependent on the renaturing solvent since it is hard to separate the folding systems from the solvent surroundings in membrane protein unfolding studies. Referee 1Technical concerns: 1. DDM CMC in ureaThe CMC of DDM in different concentrations of urea was measured and showed that micelles are present with a DDM concentration of 1mM even at the highest urea concentration of 8M (as previously used for GalP). We have clarified this in the methods (p15) and added a figure S8 to the supplementary information showing the increase in DDM CM...

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Section: Relative Stability Of Helix I and Viimentioning

confidence: 99%

Relative Domain Folding and Stability of a Membrane Transport Protein

Harris

Findlay

Simms

et al. 2014

Journal of Molecular Biology

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“…ϕ-Value analysis of the seven-transmembrane helix (TM) protein bacteriorhodopsin has now been performed twice. The initial analysis highlighted a TS with D-level compaction (17) wherein helix B (near the N terminus) has native-like contacts in the TS and thus constitutes part of the folding nucleus (18), whereas helix G (close to the C terminus) is essentially unfolded (19). It was subsequently found that bulk solution concentration of detergent can alter the folding kinetics of bacteriorhodopsin (20).…”

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Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

Paslawski

Lillelund

Kristensen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2-3 residues throughout the 178-residue transmembrane domain) of GlpG, a membraneembedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3-6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denaturedstate-level structure in the transition state. We propose that loops 1-3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated.GlpG | membrane protein | rhomboid | folding | kinetics T he biologically active structure of a protein is encoded in its sequence, and protein-folding studies aim to elucidate how this native state is reached. Great progress has been made in understanding the mechanisms of folding of water-soluble proteins based on comprehensive protein-engineering studies in combination with computational efforts (1, 2) and application of theoretical models (3-5). Much less is known about the folding mechanisms of membrane proteins that present extra challenges such as low expression levels and the need for a membrane-like environment to fold (6-11). In vivo, α-helical membrane proteins insert into the membrane cotranslationally via the signal recognition particle and Sec-translocon complex (12). Transmembrane helices exit one by one or in pairs into the lipid environment through a lateral gate in the translocon. Folding to the native state occurs spontaneously after helices are inserted into the membrane. To mimic this process, most in vitro membrane protein-folding experiments first denature the protein in SDS; renaturation is then achieved by adding excess nonionic surfactants such as dodecyl maltoside (DDM) (13).A complete protein folding mechanism must include descriptions of the denatured state (D), the native state (N), any metastable intermediates, and the transiently populated transition states (TS) that connect them. TS can only be analyzed indirectly using methods based on kinetic experiments, such as Fersht's ϕ-value approach (14, 15). The ϕ-value is the ratio between the energy perturbation to N (from equilibrium measurements or a combination of folding and unfolding kinetics) and the energy perturbation to TS (from kinetic measurements) ca...

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“…The protein solubilized by 0.4 M L-arginine and 2 M urea was refolded through stepwise dialysis and resulted in a recovery over 50% soluble protein, which was 30% higher than that of the protein solubilized without L-arginine. The more hydrophobic microenvironment of the protein solubilized with L-arginine may reduce the susceptibility to aggregation, and thus improving the protein folding [29,30].…”

Section: Discussionmentioning

confidence: 99%