Structural impact of proline mutations in the loop region of an ancestral membrane protein

Nadeau, Vincent G.; Deber, Charles M.

doi:10.1002/bip.22765

Cited by 4 publications

(2 citation statements)

References 28 publications

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“…A possible explanation for this high molecular weight band may be native oligomers or aggregates of Trx–subunit C, which did not migrate deeply into the gel, since subunit c is more prone to form aggregates when heterologously expressed 26 . These oligomeric rings and aggregates of subunit c are even resistant to boiling in SDS, especially in presence of Na + ions 27,28 . However, in comparison to subunit C without Trx‐tag, 27,28 Trx‐subunit C is largely present in a monomeric state (Figure 3b), suggesting that Trx prevents aggregation and aids in retaining subunit c as a soluble monomer.…”

Section: Resultsmentioning

confidence: 99%

“…26 These oligomeric rings and aggregates of subunit c are even resistant to boiling in SDS, especially in presence of Na + ions. 27,28 However, in comparison to subunit C without Trx-tag, 27,28 Trx-subunit C is largely present in a monomeric state (Figure 3b), suggesting that Trx prevents aggregation and aids in retaining subunit c as a soluble monomer. A strong increase in purity of Trxsubunit C as well as a loss of the band at >250 kDa occurred at temperatures above 70 C, peaking at 80 C with a purity of ~97% (Figure 3b,c).…”

Section: Resultsmentioning

confidence: 99%

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Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

et al. 2021

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Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

et al. 2021

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Evolution of Protein Physical Structures in Insect Chemosensory Systems

Picimbon

2019

Olfactory Concepts of Insect Control - Alternative to Insecticides

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Structural effects of extracellular loop mutations in CFTR helical hairpins

Chang¹,

Stone²,

Chin³

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Missense mutations constitute 40% of 2000 cystic fibrosis-phenotypic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) database, yet the precise mechanism as to how a point mutation can render the entire 1480-residue CFTR protein dysfunctional is not well-understood. Here we investigate the structural effects of two CF-phenotypic mutations - glutamic acid to glycine at position 217 (E217G) and glutamine to arginine at position 220 (Q220R) - in the extracellular (ECL2) loop region of human CFTR using helical hairpin constructs derived from transmembrane (TM) helices 3 and 4 of the first membrane domain. We systematically replaced the wild type (WT) residues E217 and Q220 with the subset of missense mutations that could arise through a single nucleotide change in their respective codons. Circular dichroism spectra of E217G revealed that a significant increase in helicity vs. WT arises in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, while this mutant showed a similar gel shift to WT on SDS-PAGE gels. In contrast, the CF-mutant Q220R showed similar helicity but an increased gel shift vs. WT. These structural variations are compared with the maturation levels of the corresponding mutant full-length CFTRs, which we found are reduced to approx. 50% for E217G and 30% for Q220R vs. WT. The overall results with CFTR hairpins illustrate the range of impacts that single mutations can evoke in intramolecular protein-protein and/or protein-lipid interactions - and the levels to which corresponding mutations in full-length CFTR may be flagged by quality control mechanisms during biosynthesis.

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Structural impact of proline mutations in the loop region of an ancestral membrane protein

Cited by 4 publications

References 28 publications

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

Heat treatment of thioredoxin fusions increases the purity of α‐helical transmembrane protein constructs

Evolution of Protein Physical Structures in Insect Chemosensory Systems

Structural effects of extracellular loop mutations in CFTR helical hairpins

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