Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase

by Baucom, Dustin R.; Furr, Mercede; Kumar, Vivek Govind; Okoto, Patience; Losey, James L.; Henry, Ralph L.; Moradi, Mahmoud; Kumar, Thallapuranam Krishnaswamy Suresh; Heyes, Colin D.

Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting com-plex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsiblefor binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsicallydisordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, andthe functional roles of its four different motifs have been debated. Using a novel combination of experimental and computationaltechniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis,site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhancedsampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agree-ment between the experimental and computational data provides a quantitatively consistent picture and allows us to identifya heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structuralensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfoldingfree energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally.We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for theLHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.

Biophysical Journal
Start Page
1542-0086; 0006-3495