Chapter 8 Methods for Measuring the Thermodynamic Stability of Membrane Proteins

Hong, Heedeok; Joh, Nathan H.; Bowie, James U.; Tamm, Lukas K.

doi:10.1016/s0076-6879(08)04208-0

Cited by 71 publications

(70 citation statements)

References 78 publications

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“…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)(4)(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)(7)(8)(9)(10)(11). In vivo, α-helical membrane proteins insert into the membrane cotranslationally via the signal recognition particle and Sec-translocon complex (12).…”

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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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confidence: 99%

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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“…To fully understand how these complexes are constructed, it is essential to measure the affinities of the interacting partners. The typical approach to measuring dissociation constants is to dilute the protein complex until it falls apart (3,4). This approach is effective for high affinity interactions in solution because it is straightforward to dilute over many orders of magnitude.…”

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Method to measure strong protein–protein interactions in lipid bilayers using a steric trap

Hong

Blois

Cao

et al. 2010

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

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178

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Measuring high affinity protein-protein interactions in membranes is extremely challenging because there are limitations to how far the interacting components can be diluted in bilayers. Here we show that a steric trap can be employed for stable membrane interactions. We couple dissociation to a competitive binding event so that dissociation can be driven by increasing the affinity or concentration of the competitor. The steric trap design used here links monovalent streptavidin binding to dissociation of biotinylated partners. Application of the steric trap method to the well-characterized glycophorin A transmembrane helix (GpATM) reveals a dimer that is dramatically stabilized by 4-5 kcal∕mol in palmitoyloleoylphosphatidylcholine bilayers compared to detergent. We also find larger effects of mutations at the dimer interface in bilayers compared to detergent suggesting that the dimer is more organized in a membrane environment. The high affinity we measure for GpATM in bilayers indicates that a membrane vesicle many orders of magnitude larger than a bacterial cell would be required to measure the dissociation constant using traditional dilution methods. Thus, steric trapping can open new biological systems to experimental scrutiny in natural bilayer environments. membrane protein | protein folding

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“…Therefore, in the absence of such sophisticated machinery, membrane proteins are extracted and purified so that biochemical, biophysical, and structural studies (including x-ray crystallography) can be performed on proteins in detergent solution (25). However, the particular composition of the lipid bilayer plays an important role in the activity of LGICs (26); removal of the lipid bilayer by detergent solubilization usually results in a decreased thermodynamic stability and sometimes in loss of functional activity (27)(28)(29).…”

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Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

Tol

Deluz

Hassaı̈ne

et al. 2013

Journal of Biological Chemistry

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Background:The 5-hydroxytryptamine receptor (5-HT 3 R) is a prototypical pentameric ligand-gated ion channel. Results: The receptor's thermal stability was investigated in native plasma membranes, in detergent solution, and in reconstituted lipid bilayers. Conclusion: Unfolding of the 5-HT 3 R occurs via hierarchical, consecutive structural transitions, which can be distinguished experimentally. Significance: Our findings serve as an important base to create receptors of increased stability for structural/functional studies.

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Chapter 8 Methods for Measuring the Thermodynamic Stability of Membrane Proteins

Cited by 71 publications

References 78 publications

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

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

Method to measure strong protein–protein interactions in lipid bilayers using a steric trap

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps*

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