Comparative Analysis of Conformational Dynamics and Nucleotide Binding in miniGαi1 and Full-Length Gαi1
- 1School of Pharmacy, Sungkyunkwan University, 2066 Seoburo, Jangan-gu, Suwon 16419, Republic of Korea
Abstract
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.
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BibTeX
@article{duan2026comparative,
title = {{Comparative Analysis of Conformational Dynamics and Nucleotide Binding in miniGαi1 and Full-Length Gαi1}},
author = {Longhan Duan and Ka Young Chung},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = nov,
doi = {10.3390/ijms27219819},
url = {https://www.mdpi.com/1422-0067/27/21/9819},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Comparative Analysis of Conformational Dynamics and Nucleotide Binding in miniGαi1 and Full-Length Gαi1
AU - Longhan Duan
AU - Ka Young Chung
PY - 2026
DA - 2026/11/01
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27219819
UR - https://www.mdpi.com/1422-0067/27/21/9819
AB - 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.
SN - 1422-0067
ER -