Membrane Proteins Can Have High Kinetic Stability

Roberts, Jefferson; Blois, Tracy M.; Bowie, James U.

doi:10.1021/ja407232b

Cited by 42 publications

(65 citation statements)

References 44 publications

(84 reference statements)

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“…Here we employed the novel steric trapping strategy developed for studying membrane protein folding 22,45 . Steric trapping couples unfolding of a doubly-biotinylated protein to competitive binding of bulky monovalent streptavidin (mSA) (Figure 4a).…”

Section: Resultsmentioning

confidence: 99%

“…Using steric trapping, k U can be determined by shifting the reaction flux dominantly towards unfolding upon addition of a molar excess of mSA-WT with a high biotin-binding affinity (Figure S5a) 45-47 . The apparent unfolding rate ( k U,app ) is asymptotic as a function of mSA concentration, the maximum value of which corresponds to k U (Figure S5b).…”

Section: Resultsmentioning

confidence: 99%

“…DGK forms a tightly packed trimer with high kinetic stability 62,63 . It does not unfold by steric trapping up to 30 days in micelles 45 , much slower than GlpG ( τ U ≈ 2 days). Thus, it is likely that the kinetic barrier of DGK unfolding is beyond the unfolding capability of FtsH.…”

Section: Discussionmentioning

confidence: 99%

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Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

et al. 2018

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ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380–550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

et al. 2018

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“…For such efforts to be extended to other classes of membrane proteins, new experimental and theoretical frameworks will be necessary. A recent development of steric trapping method that allows the reversible control of the membrane protein folding will open new opportunities for studying the forces and mechanisms of folding of membrane proteins in their native bilayer background (Blois et al 2009 ;Chang and Bowie 2014 ;Hong et al 2010 ;Hong and Bowie 2011 ;Jefferson et al 2013 ). Hansen et al 2011 ) …”

Section: Discussionmentioning

confidence: 99%

Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins

Hong

2015

Advances in Experimental Medicine and Biology

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The lipid bilayer that constitutes cell membranes imposes environmental constraints on the structure, folding and function of integral membrane proteins. The cell membrane is an enormously heterogeneous and dynamic system in its chemical composition and associated physical forces. The lipid compositions of cell membranes not only vary over the tree of life but also differ by subcellular compartments within the same organism. Even in the same subcellular compartment, the membrane composition shows strong temporal and spatial dependence on the environmental or biological cues. Hence, one may expect that the membrane protein conformations and their equilibria strongly depend on the physicochemical variables of the lipid bilayer. Contrary to this expectation, the structures of homologous membrane proteins belonging to the same family but from evolutionary distant organisms exhibit a striking similarity. Furthermore, the atomic structures of the same protein in different lipid environments are also very similar. This suggests that certain stable folds optimized for a specific function have been selected by evolution. On the other hand, there is growing evidence that, despite the overall stability of the protein folds, functions of certain membrane proteins require a particular lipid composition in the bulk bilayer or binding of specific lipid species. Here I discuss the specific and nonspecific modulation of folding, misfolding and function of membrane proteins by lipids and introduce several diseases that are caused by misfolding of membrane proteins.

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“…Among these, differential scanning calorimetry (Galisteo and Sanchez-Ruiz 1993;Sedlák et al 2014) has provided the most valuable information on the energetics of the involved processes (for a good discussion, see Sanchez-Ruiz 1992, 2010. In addition, some new approaches, such as pulse proteolysis (Di Bartolo et al 2016;Schlebach et al 2011) and steric trapping (Jefferson et al 2013), are becoming promising tools to explore complementary aspects of protein kinetic stability.…”

Section: Kinetic Stability: the Two-state Irreversible Modelmentioning

confidence: 99%

Kinetic stability of membrane proteins

2017

Biophys Rev

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Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

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Membrane Proteins Can Have High Kinetic Stability

Cited by 42 publications

References 44 publications

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins

Kinetic stability of membrane proteins

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