Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein

Schlebach, Jonathan P.; Peng, Dungeng; Kroncke, Brett M.; Mittendorf, Kathleen F.; Narayan, Malathi; Carter, Bruce D.; Sanders, Charles R.

doi:10.1021/bi301635f

Cited by 36 publications

(78 citation statements)

References 79 publications

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“…Where known, measured stabilities of helical bundle and β barrel membrane proteins are comparable to values observed for globular proteins (1.5-10 kcal/mol [15,49], corresponding to a folding equilibrium constant of ∼ 10-10 8 ). Although in a two-state folding transition, the folded and unfolded states are considered to be of fixed energies, a more realistic interpretation is to consider these as ensembles.…”

Section: B Equilibrium Properties Of the Ensemble Of Statessupporting

confidence: 58%

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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Section: B Equilibrium Properties Of the Ensemble Of Statessupporting

confidence: 58%

Model for coupled insertion and folding of membrane-spanning proteins

Hausrath

2014

Phys. Rev. E

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“…Interestingly, conformational stability measurements of diacylglycerol kinase (DAGK) (Lau & Bowie, 1997), bacteriorhodopsin (bR) (Curnow & Booth, 2007), the KcsA potassium channel (Barrera et al, 2008), and aquaglyceroporin GlpF (Veerappan et al, 2011) have suggested large thermodynamic separation of the native and denatured reference states (Δ G unf = 16 to 31 kcal/mol). On the other hand, studies of disulfide bond reducing protein B (DsbB) (Otzen, 2003), galactose transporter GalP (Findlay et al, 2010), lactose permease LacY (Harris et al, 2014), human peripheral myelin protein 22 (PMP22) (Schlebach et al, 2013), and the rhomboid protease (Baker & Urban, 2012) have suggested a more modest thermodynamic preference for their native conformations (Δ G unf = 0 to 4.5 kcal/mol). Can it be that the range of thermodynamic stabilities of single domain wild-type α-helical membrane proteins varies by more than an order of magnitude?…”

Section: Energetics Of Folding and Misfolding Of α-Helical Membranmentioning

confidence: 99%

“…Similar to soluble proteins (Brockwell & Radford, 2007), kinetic intermediates of helical membrane proteins can form within milliseconds of the initiation of folding from detergent-denatured states (Allen et al, 2004b; Booth et al, 1995; Krishnamani & Lanyi, 2011; Lu & Booth, 2000; Otzen, 2003). However, complete refolding and/or oligomerization from can require anywhere from minutes to days in vitro (Allen et al, 2004b; Cao et al, 2011; Jefferson et al, 2013; Krishnamani & Lanyi, 2011; Riley et al, 1997; Schlebach et al, 2012; Schlebach et al, 2013). The folding of the soluble denatured forms of β-barrel membrane proteins into membranes also appears to be quite slow (Burgess et al, 2008; Gessmann et al, 2014; Huysmans et al, 2010; Huysmans et al, 2012), though in this case the rate-limiting step seems to involve the transfer of the unfolded protein from the aqueous phase to the membrane phase (Gessmann et al, 2014; Huysmans et al, 2010; Huysmans et al, 2012).…”

Section: Energetics Of Folding and Misfolding Of α-Helical Membranmentioning

confidence: 99%

“…Reversible unfolding measurements of wild-type PMP22 have revealed a minimal free energy difference between folded and unfolded conformations in n -dodecylphosphocholine (DPC) micelles (Δ G unf ≈ 0 kcal/mol) (Schlebach et al, 2013) (Figure 5A). It is, of course, likely that PMP22 possesses a somewhat greater degree of conformational stability in biological membranes.…”

Section: Energetics Of Folding and Misfolding Of α-Helical Membranmentioning

confidence: 99%

“…However, a fit of the unfolding transition to a two-state equilibrium model (black line) suggests the folded conformation is still only marginally favored in the presence of glycerol (Δ G unf = 1.5 ± 0.1 kcal/mol). (Figure from Schlebach et al 2013) B) The structural dynamics of WT and L16P PMP22 were assessed in tetradecylphosphocholine (TDPC) micelles using solution NMR and other methods. The results indicate that the initial state of PMP22 unfolding involves dissociation of its first transmembrane segment from the 4-helix bundle, with the remaining 3-helix bundle adopting a molten globule-like state in the membrane.…”

Section: Figurementioning

confidence: 99%

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The safety dance: biophysics of membrane protein folding and misfolding in a cellular context

Schlebach

Sanders

2014

Quart. Rev. Biophys.

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Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine.

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Unfolding study of a trimeric membrane protein AcrB

Cui

Wang

et al. 2014

Protein Science

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The folding of a multi-domain trimeric a-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrB Dloop , from the SDS unfolded state. The CD spectrum of the refolded AcrB Dloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB.

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Reversible Folding of Human Peripheral Myelin Protein 22, a Tetraspan Membrane Protein

Cited by 36 publications

References 79 publications

Model for coupled insertion and folding of membrane-spanning proteins

Model for coupled insertion and folding of membrane-spanning proteins

The safety dance: biophysics of membrane protein folding and misfolding in a cellular context

Unfolding study of a trimeric membrane protein AcrB

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