Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster H17 - Interpreting Crowding Effects on FRET Signals for Protein Kinetics Analysis

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Traditional methods for simulating cellular environments in enzymatic studies often involve observing reactions in dilute buffers. However, the intracellular space is densely packed with macromolecules such as proteins, nucleic acids, ribonucleoproteins, polysaccharides, and metabolites, creating a highly crowded environment. This phenomenon, known as crowding, significantly affects enzymatic activities both in vitro and in vivo, thereby challenging the validity and accuracy of dilute buffer models. This study examines how crowding influences the dissociation constant (KD) values through quantitative Förster resonance energy transfer (qFRET) imaging. Bovine serum albumin (BSA) serves as a crowding agent to mimic cellular conditions, and qFRET technology interprets FRET signals to quantify binding kinetics. An in vitro assay was conducted using a 96-well plate with varying BSA concentrations to simulate different levels of crowding and assess their impact on KD values. FRET measurements in solution phase were performed to replicate protein interaction affinities in living cells. The study focuses on the reversible SUMOylation reaction between a small ubiquitin-like modifier (SUMO) and its E2 ligase, Ubc9, using fluorescent proteins CyPet-SUMO1 and YPet-Ubc9 to determine KD. The dissociation constant, KD = [A][B]/[AB], where [A] is CyPet-SUMO1, [B] is YPet-Ubc9, and [AB] is the concentration of the complex, reflects the ratio of free to bound proteins. A one-way ANOVA will evaluate the statistical significance of KD variations between crowded and non-crowded conditions. This research provides insights into protein interactions and cellular crowding, with potential implications for pharmaceuticals, bioseparations processes, and drug discovery targeting protein-protein interactions.

Materials and

Methods:  The experimental setup for finding absolute FRET (EmFRET) signals utilizes a Costar® black and clear-bottom 96 well-plate, a solution of bovine serum albumin (BSA) at 350 g/L with a pH of 7.94, and engineered fluorescent proteins CyPet-SUMO1 and YPet-Ubc9. 350 g/L of BSA solution was diluted with artificial cerebrospinal fluid (aCSF) salt solution to achieve various concentrations for FRET readings. Through experimental testing, a low-crowded environment was selected at 95 g/L of BSA, and a high-crowded environment was selected at 290 g/L of BSA. CyPet-SUMO1 (55.50 µM) and its E2 ligase, YPet-Ubc9 (61.1 µM), were obtained through protein purification. To determine the necessary volume of BSA for each well, calculations were performed using 1 µM of CyPet and YPet to achieve the target concentrations, ensuring each well contained 200 µL of total suspended solution. These proteins were incubated at 37°C to simulate cellular conditions, followed by homogenization and spectrophotometric analysis using a SpectraMax M3™. The spectrophotometer measured emission wavelengths from 414 nm to 530 nm, and excited at 414 nm and 475 nm. BSA signals were measured separately and subtracted to ensure accurate protein affinity measurements on the EmFRET readings. After obtaining EmFRET readings, KD was determined for non-crowding (0 g/L BSA), low-crowding (95 g/L BSA), and high-crowding (290 g/L BSA) conditions. SoftMaxPro software facilitated data collection, and Prism 5 software was used for KD calculations. These calculations used equations involving fixed CyPet-SUMO1 concentrations, varied YPet-Ubc9 concentrations, and maximum EmFRET signals.

Results, Conclusions, and Discussions: EmFRET signals were assessed using BSA concentrations ranging from 70-100 g/L to determine the optimal level for simulating a low-crowded environment, and from 250-300 g/L to represent high-crowded conditions. After evaluating these ranges and conducting three trials to minimize inaccuracies, 290 g/L of BSA was selected for high-crowded conditions, and 95 g/L for low-crowded conditions in determining KD. Additionally, a control group with no BSA was included to provide a baseline for comparison in a non-crowded environment. The FRET signals displayed expected patterns for CyPet-SUMO1 and YPet-Ubc9, with CyPet emitting fluorescence from 414 nm to 475 nm, and YPet from 475 nm to 530 nm. In this system, CyPet undergoes quenching (loses energy), while YPet experiences excitation (gains energy). KD and maximum EmFRET signals were calculated using nonlinear regression analysis for each experiment with varying total concentrations of YPet-Ubc9 (μM). The KD value for CyPet-SUMO1 and YPet-Ubc9 was determined from the relationship between EmFRET (RFU) and [YPet-Ubc9]total (μM) and the analysis revealed KD values of 3.5 ± 0.5 μM, 12.4 ± 3.7 μM, and 10.6 ± 2.5 μM for 0, 95, and 290 g/L of BSA, respectively. The data indicates that the crowding agent elevates KD by approximately a factor of three, underscoring the significant impact of crowding on KD. This indicates that in crowded conditions, additional molecules (crowders) reduce protein affinity, leading to weaker binding compared to non-crowded environments or diluted solutions. This challenges traditional approaches that use dilute buffers to mimic cellular environments for studying enzymatic processes. The ANOVA test showed p-values of 0.0123 for non-crowded vs. low-crowded and 0.0340 for non-crowded vs. high-crowded environments, both below 0.05, indicating significant differences. This suggests sufficient evidence to reject the null hypothesis and conclude significant differences exist between non-crowded and crowded environments. However, the Brown-Forsythe test revealed no significant difference between high-crowding and low-crowding conditions (p = 0.3891), suggesting that varying BSA concentrations (95 g/L to 290 g/L) do not significantly affect KD values. Significant differences were observed between non-crowded and crowded environments. EmFRET signals were noisier at 290 g/L of BSA, indicating that crowding may interfere with spectrometer signals.

Acknowledgements (Optional):  I am deeply grateful to Dr. Victor G. J. Rodgers for his unwavering support, encouragement, and belief in me. His mentorship has been invaluable, providing not only academic guidance but also genuine care and dedication to my growth as a researcher, scientist, and confident individual. I also extend my sincere thanks to Dr. Jiayu Liao for granting me access to his lab and offering crucial guidance and support for my research. Working with his exceptional graduate students, Chuchu Liu and Gagan Mannur, has been a privilege. Additionally, I express gratitude to the Biotransport and Bioreaction Kinetics (B2K) Group.