The Library
Bacterial expression, purification and biophysical characterization of the smallest plant reticulon isoform, RTNLB13
Tools
Tools
Tools
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
|
PDF
WRAP-Bacterial-expession-purification-biophysical-Frigerio-2018.pdf - Accepted Version - Requires a PDF viewer. Available under License Creative Commons Attribution Non-commercial No Derivatives 4.0. Download (3524Kb) | Preview |
Official URL: http://dx.doi.org/10.1016/j.pep.2018.06.015
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: |
|
||||||||
| 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 | ||||||||
| RIOXX Funder/Project Grant: |
|
Request changes or add full text files to a record
Repository staff actions (login required)
 |
View Item |
Downloads
Downloads per month over past year
