Out but Not In: The Large Transmembrane β-Barrel Protein FhuA Unfolds but Cannot Refold via β-Hairpins

Thoma, Johannes; Bosshart, Patrick D.; Pfreundschuh, Moritz; Müller, Daniel J.

doi:10.1016/j.str.2012.10.006

Cited by 46 publications

(67 citation statements)

References 33 publications

(62 reference statements)

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“…Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20)(21)(22)(23)(24)(25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26-35).…”

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confidence: 99%

“…Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20)(21)(22)(23)(24)(25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26)(27)(28)(29)(30)(31)(32)(33)(34)(35).…”

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confidence: 99%

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Substrate-induced changes in the structural properties of LacY

Serdiuk

Madej²,

Sugihara³

et al. 2014

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

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The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugarand H + -binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H + -and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H + symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.atomic force microscopy | membrane | transport protein | membrane protein structure | membrane protein folding | membrane transport T he lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H + (sugar/H + symport) (3-6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H + with the electrochemical H + gradient (Δμ H + ; interior negative and/or alkaline). Conversely, because coupling between sugar and H + is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H + transport with the generation of Δμ H +, the polarity of which depends upon the direction of the sugar concentration gradient (7-10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13-16). At the apex of the h...

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confidence: 99%

mentioning

confidence: 99%

Substrate-induced changes in the structural properties of LacY

Serdiuk

Madej²,

Sugihara³

et al. 2014

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

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“…While the refolding pathway of chemically denatured OmpA protein appears to be best described by the concerted process, it is interesting to note that mechanical unfolding of the related protein KpOmpA from Klebsiella pneumoniae results in a stepwise unfolding process where individual β hairpins unfold successively [64]. Similar behavior has also been observed in β barrels OmpG and FhuA [65,66]. Moreover, in the case of OmpG, refolding was also observed and occurred in a stepwise fashion involving insertion of individual β hairpins [65].…”

Section: Folding Pathways For β Barrelsmentioning

confidence: 76%

Model for coupled insertion and folding of membrane-spanning proteins

Hausrath

2014

Phys. Rev. E

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Current understanding of the forces directing the folding of integral membrane proteins is very limited compared to the detailed picture available for water-soluble proteins. While mechanistic studies of the folding process in vitro have been conducted for only a small number of membrane proteins, the available evidence indicates that their folding process is thermodynamically driven like that of soluble proteins. In vivo, however, the majority of integral membrane proteins are installed in membranes by dedicated machinery, suggesting that the cellular systems may act to facilitate and regulate the spontaneous physical process of folding. Both the in vitro folding process and the in vivo pathway must navigate an energy landscape dominated by the energetically favorable burial of hydrophobic segments in the membrane interior and the opposition to folding due to the need for passage of polar segments across the membrane. This manuscript describes a simple, exactly solvable model which incorporates these essential features of membrane protein folding. The model is used to compare the folding time under conditions which depict both the in vitro and in vivo pathways. It is proposed that the cellular complexes responsible for insertion of membrane proteins act by lowering the energy barrier for passage of polar regions through the membrane, thereby allowing the chain to more rapidly achieve the folded state.

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“…In 2009, Sapra et al [59] investigated the unfolding of the membrane protein OmpG that was reconstituted in lipids and revealed that the unfolding pathway was via amino acids 8,43,83,126,166,204, and 248. In 2012, Thoma et al [65] investigated the unfolding of membrane protein FhuA: after attaching the E. coli polar lipid bilayers containing reconstituted FhuA molecules onto freshly cleaved mica, AFM imaging was first performed to acquire the morphology of FhuA (Fig. 3f, g), and then SMFS was performed to mechanically unfold single FhuA molecules, showing that the unfolding pathway of FhuA was amino acid 92, 161,192,229,297,367,447,492,536,584,634,681 (Fig.…”

Section: Mechanically Unfolding Membrane Proteins With Smfsmentioning

confidence: 99%

Progress in measuring biophysical properties of membrane proteins with AFM single-molecule force spectroscopy

Liu

et al. 2014

Chin. Sci. Bull.

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Membrane proteins are crucial in cell physiological activities and are the targets for most drugs. Thus, investigating the behaviors of membrane proteins not only provide deeper insights into cell function, but also help disease treatment and drug development. Atomic force microscopy is a unique tool for investigating the structure of membrane proteins. It can both image the morphology of single native membrane proteins with high resolution and, via single-molecule force spectroscopy (SMFS), directly measure their biophysical properties during molecular physiological activities such as ligand binding and protein unfolding. In the context of molecular biomechanics, SMFS has been successfully used to understand the structure and function of membrane proteins, complementing the static three-dimensional structures of proteins obtained by X-ray crystallography. Here, based on the authors' antigen-antibody binding force measurements in clinical tumor cells, the principle and method of SMFS is discussed, the progress in using SMFS to characterize membrane proteins is summarized, and challenges for SMFS are presented.

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Out but Not In: The Large Transmembrane β-Barrel Protein FhuA Unfolds but Cannot Refold via β-Hairpins

Cited by 46 publications

References 33 publications

Substrate-induced changes in the structural properties of LacY

Substrate-induced changes in the structural properties of LacY

Model for coupled insertion and folding of membrane-spanning proteins

Progress in measuring biophysical properties of membrane proteins with AFM single-molecule force spectroscopy

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