Comparative Analysis of Conformational Dynamics and Nucleotide Binding in miniGαi1 and Full-Length Gαi1

Mini G proteins (miniG proteins) are engineered Gα constructs lacking the α-helical domain (AHD) and are widely used to stabilize active G protein-coupled receptor (GPCR) conformations for structural and biochemical studies. However, the intrinsic conformational and nucleotide-binding properties of miniG proteins remain incompletely understood relative to their full-length counterparts. Here, we compared miniGαi1 and full-length Gαi1 using fluorescence-based nucleotide-binding assays and hydrogen–deuterium exchange mass spectrometry (HDX-MS). BODIPY–FL–GTP assays showed that miniGαi1 interacted with GTP more rapidly and produced higher fluorescence intensity than full-length Gαi1. Unlike full-length Gαi1, miniGαi1 did not exhibit fluorescence decay following nucleotide binding, suggesting impaired or absent GTP hydrolysis activity. HDX-MS analysis demonstrated globally higher deuterium uptake in miniGαi1, particularly in regions associated with nucleotide binding and GPCR interaction. These results indicate that miniGαi1 adopts a more solvent-accessible and conformationally dynamic state than GDP-bound full-length Gαi1. Together, our findings provide biochemical and structural insight into the intrinsic properties of miniG proteins.