Science and Technology Production
Reunión Conjunta de Sociedades de Biociencias - EVALUATION OF THE CONFORMATIONAL CHARACTERISTICS OF THE HUMAN LYR PROTEIN ISD11.
Congress
Date:
2017Publishing House and Editing Place:
Reunión Conjunta de Sociedades de BiocienciasSummary *
ISD11 is a small protein that interacts with the cysteine desulfurase NFS1, a key-component of the Fe-S cluster biogenesis machinery inside the mitochondria. This interaction provides stability to NFS1 protein complex. In addition, the conserved LYR sequence of ISD11 has a central role in the interaction with ISCU/HSPA9/HSC20 protein complex that transfers Fe-S clusters from ISCU to target proteins. Recently, it was shown that ISD11 also interacts with the mitochondrial acyl carrier protein (ACP). As previously predicted, it was found that ISD11 exhibits alpha helical structure. More important, the structure shows interactions between the acyl chain (linked via phosphopantetheine cofactor to ACP) and ISD11 core residues in addition to electrostatic interactions between both proteins. In this context, our main objective is to understand how ISD11 acquires its conformation and stability. Results showed that purified (isolated) ISD11 is completely insoluble, it has a high tendency to aggregate and standard refolding experiments fail. However, we found that the addition of the detergent n-Dodecyl β-D-maltoside (DDM) and high concentration of NaCl during refolding allows us to obtain soluble protein with high secondary structure content, as judged by Far-UV CD spectra analysis. We conjecture that the detergent may mimic the acyl group and its structural role, whereas NaCl diminishes the electrostatic repulsion that takes place on the surface of ISD11 when protein partners are absent. This result open the possibility to study the role of the acyl chain on ISD11 conformational stability and the role of ISD11 as a hub protein in the interactions with NFS1 and ACP among others.To further evaluate ISD11 conformation and due to its insolubility, we also decided to obtain the helical regions of ISD11 as three peptides and evaluate the secondary structure and activity of these regions in the context of the NFS11-ISD11 complex. By Far-UV Circular Dichroism spectra we have confirmed the alpha-helix conformation of this regions and we have determined that the N terminal region is able to interact with negative charged membranes. This suggest that SD11 N-terminal regions will be important in the anchoring of the Fe-S cluster formation in the mitochondrial membrane. Information provided by the agent in SIGEVAKey Words
LYR PROTEIN FAMILYDESULFURASE ACTIVITYSTRUCTURAL CHARACTERIZATION