Probing Ca2+-Binding Capability of Viral Proteins with the EF-Hand Motif by Grafting Approach
Ca2+ is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca2+-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca2+ competition assay for experimental verification of predicted Ca2+-binding sites. This protocol will be valuable for the prediction and identification of unknown Ca2+-binding sites in virus.
Key wordsCalcium CD2 EF-hand Luminescence resonance energy transfer Protein engineering Protein–ligand interaction Terbium Virus
We appreciate the critical review by Natalie White. This work is supported in part by research from NIH GM081749 to Jenny J. Yang.