Integrating Fluorescence and Calorimetry to Teach Protein Unfolding Thermodynamics: A Collaborative Laboratory Experiment

High Resolution Image Download MS PowerPoint Slide This laboratory experiment integrates fluorescence spectroscopy and differential scanning calorimetry (DSC) to teach the thermodynamics of protein thermal unfolding through a collaborative, guided inquiry approach. Using lysozyme as a model system, students determine the melting temperature ( T m ), enthalpy (Δ H ), entropy (Δ S ), and heat-capacity change (Δ C p ) associated with thermal unfolding. The activity combines complementary techniques and analytical methods: groups analyze fluorescence data using three alternative van’t Hoff approaches (linear analysis, derivative method, and nonlinear global fitting), while others perform DSC measurements under different heating rates, protein concentrations, and reversibility conditions. All data sets are processed and fitted using an affordable, cross-platform data analysis program, providing an accessible alternative to proprietary software supplied with commercial DSC instruments. Students appropriately explore the relationships among equilibrium, reversibility, and kinetic effects by comparing fluorescence- and calorimetry-derived thermodynamic parameters within the same two-state model. The collaborative, guided inquiry design promotes a deeper conceptual understanding of protein stability and equilibrium thermodynamics while fostering transferable skills in data modeling, teamwork, and scientific communication. This experiment is suitable for upper-level undergraduate and graduate laboratory courses in physical chemistry, thermal analysis, and biochemistry.