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Bacterial expression, purification and biophysical characterization of the smallest plant reticulon isoform, RTNLB13

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Chow, Michael, Sklepari, Meropi, Frigerio, Lorenzo and Dixon, Ann M. (2018) Bacterial expression, purification and biophysical characterization of the smallest plant reticulon isoform, RTNLB13. Protein Expression and Purification, 152 . pp. 31-39. doi:10.1016/j.pep.2018.06.015 ISSN 1046-5928.

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Official URL: http://dx.doi.org/10.1016/j.pep.2018.06.015

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Abstract

Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved. We believe that this is, in part, due to difficulties in obtaining reticulon proteins in yields sufficient for structural study. To address this, we report here the first bacterial overexpression, purification, and biophysical investigation of a reticulon protein from plants, the RTNLB13 protein from A. thaliana. RTNLB13 is the smallest plant reticulon and is made up of a single RHD. We used circular dichroism, SDS-PAGE and analytical ultracentrifugation to reveal that RTNLB13 is 45% α-helical in a number of detergent environments, monomeric at low concentrations, and capable of self-association at higher concentrations. We used solution-state NMR to screen the effect of detergent type on the fold of isotopically-enriched RTNLB13, and found that ∼60% of the expected protein peaks were broadened due to slow dynamics. This broadening points toward a large network of protein-membrane interactions throughout the sequence. We have interpreted our results in light of current literature and suggest a preliminary description of RTNLB13 structure and topology.

Item Type: Journal Article
Subjects: Q Science > QK Botany
Divisions: Faculty of Science, Engineering and Medicine > Science > Chemistry
Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH): Endoplasmic reticulum, Plant proteins, Circular dichroism, Nuclear magnetic resonance spectroscopy, Arabidopsis thaliana
Journal or Publication Title: Protein Expression and Purification
Publisher: Elsevier
ISSN: 1046-5928
Official Date: December 2018
Dates:
DateEvent
December 2018Published
30 June 2018Available
29 June 2018Accepted
Volume: 152
Page Range: pp. 31-39
DOI: 10.1016/j.pep.2018.06.015
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Restricted or Subscription Access
Date of first compliant deposit: 3 July 2018
Date of first compliant Open Access: 30 June 2019
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
EP/F500378/1[EPSRC] Engineering and Physical Sciences Research Councilhttp://dx.doi.org/10.13039/501100000266

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