Expression, purification, experimental and computational biophysical characterization of SbRUVBL1/2 protein

ATPases belong to the AAA+ superfamily are associated with diverse cellular activities. The structure of members of this superfamily is mainly composed by nucleotide-binding domains (NBD), which has typical motives known as Walker A and Walker B. AAA+ proteins have a wide spectrum of functions, from DNA replication to protein quality control. RUVBL1/2 from Sorghum bicolor (SbRUVBL1/2) is a complex that is part of a molecular machine named R2TP fromthe first identification with proteins from yeast (Rvb1-Rvb2-Tah1-Pih1). Due to the wide spectrum of functions, it is of utmost importance to characterize the structure and function of the complex. This work started with the identification of the primary sequence of the proteins and the search for high similar identity primary sequence. Based on PSIPRED prediction of the primary sequence, SbRUVBL1 has 43% and 19% of alpha helices and beta sheets, while SbRUVBL2 has 41% and 20%, respectivelly. The most similar sequences are from Panicum hallii and Zea mays, sharing ~97% of similarity. The structures of SbRUVBL1/2 were modelled with the deep-learning–based structure prediction methods, RoseTTaFold and AlphaFold2. Both structures agreed with a good RMSD of ~1.7 Å, their stereochemical quality were verified by Ramachandran plot. The sequence of proteins SbRUVBL1/2 were cloned into a pETDuet vector and co-expressed in E. coli. The protein complex was purified to a high purity level (>95%) with nickel affinity cromatography followed by size exclusion cromatography. The conformation was characterized using circular dichroism spectropolarimetry, whose results agreed with the theoretical prediction based on the proteins primary sequence.