Biophysical characterization of an engineered recombinant Zeocin Binding Protein (ZBP) mutant.

Traditional strategies for identifying molecular chaperones are often indirect and nonspecific, such as monitoring stress-induced changes in gene expression or assessing in vitro chaperone activity, which are labor-intensive and may yield false-negative results. To overcome these challenges, we engineered a Zeocin Binding Protein (ZBP) variant, P9E, as a biosensor for protein folding and stability in vivo. ZBP naturally binds Zeocin, neutralizing its toxicity, and exhibits high solubility and stability. In contrast, the ZBP P9E mutant exhibits a dramatic reduction in this protective function: cells expressing ZBP P9E display approximately a 100,000-fold decrease in Zeocin resistance at 1000 μg/ml compared to cells expressing ZBP wild type, indicating severe loss of function due to altered folding and stability. In this study, ZBP P9E was recombinantly produced in E.coli, purified by single-step Ni-NTA chromatography and characterized using biophysical techniques. Thermal unfolding experiments showed that the P9E mutant has ∼26 °C lower stability than wild type, and spectroscopic analyses revealed rapid loss of secondary structure with heating, transitioning to a random coil above 30 °C. These characteristics make ZBP P9E a highly sensitive in vivo reporter of protein folding status. Beyond chaperone discovery, the ZBP P9E biosensor has broad potential to monitor proteostasis, screen folding modulators, and study protein stability dynamics in both prokaryotic and eukaryotic systems.