Monitoring Protein Deamidation in Real Time Using NMR

Abstract

Deamidation, a modification of glutamine residues in host proteins, plays a key role in bacterial pathogenesis, where bacterial effectors manipulate host signaling pathways by modifying proteins like ubiquitin (Ub) and Ub-like protein NEDD8. The study uses traditional and real-time NMR-based techniques to investigate the enzymatic activity of two bacterial deamidases, cycle inhibitory factors, CIFEC from Escherichia coli and CIFBP from Burkholderia pseudomallei. We employed BEST-HSQC NMR spectroscopy to monitor real-time deamidation of ubiquitin, providing a robust and efficient method for quantifying enzyme activity. Our findings highlight significant differences in catalytic efficiency between CIFEC and CIFBP, despite their structural similarities. NMR-based rate measurements show CIFBP has higher catalytic efficiency than CIFEC, consistent with the previous reports, while kinetic analysis of CIFEC indicates relatively weak substrate binding and suboptimal efficiency, suggesting a potential regulatory role during infections. While the overall globular fold of the ubiquitin remains unchanged upon deamidation, we observed changes in the local chemical environment, suggesting potential localized structural changes. This study extends the use of NMR spectroscopy to investigate irreversible posttranslational modifications (PTMs) like deamidation, offering a valuable tool for understanding the molecular mechanisms behind bacterial manipulation of host cellular processes.