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Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes

Jacopo Marino1, Natalie Bordag, Sandro Keller

  • 1Department of Chemistry, University of Zürich, Switzerland.

Protein Science : a Publication of the Protein Society
|October 10, 2014
PubMed

Insights

Investigating the Bacillus subtilis protein Mistic, this study found that three of its four helical fragments interact with bacterial membranes. These fragments can facilitate the overexpression of other membrane proteins, impacting quantity and quality.

Area of Science:

  • Membrane protein biology
  • Bacterial protein interactions
  • Structural biology

Background:

  • The Bacillus subtilis protein Mistic's membrane interaction and role in protein overexpression are debated.
  • Understanding Mistic's function requires dissecting its interaction with bacterial membranes.

Purpose of the Study:

  • To determine if individual helical fragments of Mistic interact with membranes in vivo and in vitro.
  • To assess the ability of Mistic fragments to promote membrane protein overexpression.

Main Methods:

  • Production of GFP-fused Mistic helical fragments for cellular localization studies in Escherichia coli.
  • Solid-phase peptide synthesis of Mistic helical fragments and circular dichroism spectroscopy to study secondary structure acquisition in lipids and detergents.
  • Bioinformatic analysis of the Mistic protein family.

Main Results:

  • The third helical fragment of Mistic interacts with LDAO micelles but not lipid bilayers.
  • The other three helical fragments interact with membranes both in vivo and in vitro.
  • All helical fragments, when fused to an N-terminal sequence, can replace full-length Mistic for G-protein-coupled receptor overexpression in E. coli, with varying outcomes.
  • Bioinformatic analysis identified 20 Mistic homologs and a conserved Shine-Dalgarno sequence crucial for downstream translation.

Conclusions:

  • Specific helical fragments of Mistic mediate membrane interactions.
  • Mistic fragments can functionally replace the full-length protein in promoting membrane protein overexpression.
  • A conserved Shine-Dalgarno sequence is important for the translation of Mistic-associated genes.
Keywords:
GFP-fusionGPCR expressionMisticlipidspeptide-membrane interactionyugO

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