Mapping the energy landscape for second-stage folding of a single membrane protein

Min, Duyoung; Roberts, Jefferson; Bowie, James U.; Yoon, Tae–Young

doi:10.1038/nchembio.1939

Cited by 76 publications

(110 citation statements)

References 60 publications

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“…S29). Because Ail is a kinetically stabilized barrel, this kinetic partitioning regulates barrel stability and aggregation propensity through a mechanism similar to how amyloids are formed (30,31). We conclude that the cooperativity and the rate of aggregation dictate the fate of the barrel, with our results confirming the importance of the kinetic component on the stabilization of folded Ail.…”

Section: Kinetic Partitioning In Omp Stability and Aggregationsupporting

confidence: 71%

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Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation

Gupta

Mahalakshmi

2019

J. Biol. Chem.

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Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans.

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Section: Kinetic Partitioning In Omp Stability and Aggregationsupporting

confidence: 71%

“…S1). Additionally, kinetically stabilized protein structures (such as Ail) are both sensitive to mutational effects and are implicated in protein misfolding diseases (2,6,11,30).…”

Section: Kinetic Partitioning In Omp Stability and Aggregationmentioning

confidence: 99%

Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation

Gupta

Mahalakshmi

2019

J. Biol. Chem.

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“…Previous studies indicate that GFP that has been transiently unfolded by ClpXP can fold back to the native state when the ATP hydrolysis rate is low 58,59 . The k F 's of Barnase and DHFR in water, which cannot be degraded by FtsH, are ∼300 and ∼8 fold higher ( k F ≈ 12 s -1 and 0.25 s -1 ), respectively, than that of GlpG in neutral bicelles ( k F, ≈ 0.04 s -1 ) 32,60,61 . Notably, unfolded DHFR can rapidly refold to a compact intermediate with k F,U-I ≈ 6.7 s -1 60 .…”

Section: Discussionmentioning

confidence: 93%

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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“…An AFM experiment on an α-helical membrane protein, antiporter (N ≈ 380), whose γ value we find is ≈ 1.1, could not be refolded to the original form after mechanically unfolded [70]. However, a recent remarkable single molecule force experiment [71] has shown that GlpG, an α-membrane proteins with N ≈ 270, can reversibly fold in bicelles even after the entire structure including TM helices is disrupted by mechanical forces. Remarkably, we find γ ≈ 1.5 for GlpG.…”

Section: Discussionmentioning

confidence: 95%

Contact Statistics Highlight Distinct Organizing Principles of Proteins and RNA

Liu

Hyeon

2016

Biophysical Journal

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Although both RNA and proteins have densely packed native structures, chain organizations of these two biopolymers are fundamentally different. Motivated by the recent discoveries in chromatin folding that interphase chromosomes have territorial organization with signatures pointing to metastability, we analyzed the biomolecular structures deposited in the Protein Data Bank and found that the intrachain contact probabilities, P(s) as a function of the arc length s, decay in power-law ∼s(-γ) over the intermediate range of s, 10 ≲ s ≲ 110. We found that the contact probability scaling exponent is γ ≈ 1.11 for large RNA (N > 110), γ ≈ 1.41 for small-sized RNA (N < 110), and γ ≈ 1.65 for proteins. Given that Gaussian statistics is expected for a fully equilibrated chain in polymer melts, the deviation of γ-value from γ = 1.5 for the subchains of large RNA in the native state suggests that the chain configuration of RNA is not fully equilibrated. It is visually clear that folded structures of large-sized RNA (N ≳ 110) adopt crumpled structures, partitioned into modular multidomains assembled by proximal sequences along the chain, whereas the polypeptide chain of folded proteins looks better mixed with the rest of the structure. Our finding of γ ≈ 1 for large RNA might be an ineluctable consequence of the hierarchical ordering of the secondary to tertiary elements in the folding process.

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Mapping the energy landscape for second-stage folding of a single membrane protein

Cited by 76 publications

References 60 publications

Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation

Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation

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

Contact Statistics Highlight Distinct Organizing Principles of Proteins and RNA

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