Zhilin Luo
M.R. Bauer Foundation Summer Science Research Fellow
Street Lab, Department of Biochemistry
Brandeis University
Co-Chaperone Mechanism of J-Domain Protein and BiP
ERdj3 (JD) and BiP are endoplasmic reticulum (ER) molecular chaperones that play critical roles in protein folding. Much qualitative evidence indicates that these two chaperones can coordinate their functions, but the underlying mechanism is unclear. A traditional model proposed that JD will accelerate the ATPase activity of BiP, but it only predicts JD’s catalytical function in the forward reaction—ATP hydrolysis. Nonetheless, experimental data shows that JD promotes the reverse reaction as well. Meanwhile, the old model failed to interpret some of the observations we had in the Fluorescence Resonance Energy Transfer (FRET) and ATPase activity assays. A new model, therefore, is constructed, introducing a potential intermediate within the ATPase activity cycle. With this new model, most of the unresolved questions are addressed, which includes the discrepancy between the apparent binding constant obtained from ATPase assay and true binding constant derived from Fluorescent Polarization (FP), the bump observed in the FRET ATP flush experiment, and JD’s catalytical function in the reverse reaction. To validify this new model, however, a more advanced yet complicated experiment called Single Molecule FRET (SM-FRET) is required. Such technique enables us to directly determine the existence of the intermediate as it only measures one BiP molecule, and any conformational changes within the reaction cycle would be recorded. Future work, consequently, will primarily focus on preparing and conducting the SM-FRET and adjust our new model based on the results obtained.Personal Statement and Pandemic Reflection
Working in Street Lab has been an extremely fruitful and exciting experience to me even though under the circumstance of the global pandemic. I was lucky because the university has lifted all the restrictions from the COVID so that I could get back into lab and work nearly full time. Despite this, the pandemic had still influenced my pace as people were still paying attention to the social distance, and some of the cooperative works were just hard to proceed as normal. For instance, graduate students in our lab used to work full time and were able to train our undergrads in person, yet under the current circumstance, people tended to reserve for the equipment ahead and avoid being in touch with one another. This situation could become annoying when I was trying to learn new techniques. With the skills collected from previous semesters and the help from the people in lab, however, I was able to make thrilling progress on my project.
The major advance I made on the project was on building models to explain the data collected by the previous researchers in lab and conducting further experiments to validify the model. One unique part of my research was that it contained only the works from undergrads, and I was the fourth undergrad who tried to push this project forward. Therefore, before I even started to work on this project, a lot of data had been taken, and consequently, a critical part of my goal was to put all these puzzles into one piece. An old model constructed in an earlier time failed to explain the discrepancy between the apparent binding constant and true binding constant, and it also did not provide a good rationale on why J-domain also helped to accelerate the reverse reaction of BiP hydrolysis cycle. This made me realize that the actual mechanism underlying might be much more complicated than this. With the great support from Prof. Street, I was able to come up with a more reasonable model after months of thinking and testing process. Theoretically, with the new model, we were able to explain the discrepancies observed, yet to prove the eligibility of our model, we need to conduct Single Molecule FRET (SM-FRET) experiment. As a highly specialized experiment, a series of preparation was required, which could take up to a few months. Meanwhile, Prof. Street pointed out that some characteristics we saw in our FRET data resembled that of the experiment with peptides such as CH1 peptide. In all, this summer has been an exciting and fruitful one to me, and by working with data collected from different people and putting them into one piece, I was able to strengthen my skill of problem finding and solving. Lastly, I would like to share my gratitude to Bauer Foundation again for providing the fund.