Dataset Information

Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

ABSTRACT: BACKGROUND: The Arabidopsis thaliana protein atTic20 is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. As a component of the TIC complex, it is believed to form a preprotein-conducting channel across the inner membrane. RESULTS: We report a method for producing large amounts of recombinant atTic20 using a codon-optimized strain of E. coli coupled with an autoinduction method of protein expression. This method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes. Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain. CONCLUSION: Our work strengthens the hypothesis that atTic20 functions similarly to other small ?-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.

SUBMITTER: Campbell JH

PROVIDER: S-EPMC4307631 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

Campbell James H JH Hoang Tuan T Jelokhani-Niaraki Masoud M Smith Matthew D MD

BMC biochemistry 20141231

<h4>Background</h4>The Arabidopsis thaliana protein atTic20 is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. As a component of the TIC complex, it is believed to form a preprotein-conducting channel across the inner membrane.<h4>Results</h4>We report a method for producing large amounts of recombinant atTic20 using a codon-optimized strain of E. coli coupled with an autoinduction method of protein expression. This method resulted in the recombina ...[more]

PMID: 25551276

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Project description:All-atom (AA) molecular dynamics simulations are used to unravel the binding mechanism of yeast oxysterol binding protein (Osh4) to model membranes with varying anionic lipid concentration using AA and the highly mobile membrane mimetic (HMMM) representations. For certain protein-lipid interactions, an improved forcefield description is used (CUFIX) to accurately describe lipid-protein electrostatic interactions. Our detailed computational studies have identified a single, β-crease oriented, membrane-bound conformation of Osh4 for all anionic membranes. The penetration of the PHE-239 residue below the membrane phosphate plane is the characteristic signature of the membrane-bound state of Osh4. As the phenylalanine loop anchors itself deeply in the membrane; the other regions of the Osh4, namely, ALPS motif, β6- β7 loop, β14- β15 loop, and β16- β17 loop, maximize their contact with the membrane. Furthermore, loose lipid packing and higher mobility of HMMM enable stronger association of the ALPS motif with the membrane lipids through its hydrophobic surface. After the HMMM is converted to AA and equilibrated, the binding is two to three times stronger compared with simulations started with the AA representation, yielding the major importance of the ALPS motif to binding. Quantitative estimation of binding energy revealed that the phenylalanine loop plays a crucial role in stable membrane attachment of Osh4 and contributes significantly toward overall binding process. The CUFIX parameters provide a more balanced picture of hydrophobic and electrostatic interactions between the protein and the membrane, which differs from our past work that showed salt bridges alone stabilized Osh4-membrane contact. Our study provides a comprehensive picture of the binding mechanism of Osh4 with model single membranes and, thus, understanding of the initial interactions is important for elucidating the biological function of this protein to shuttle lipids between organelles.

Dataset Information

Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

Publications

Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

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