Structural Characterization and Comparison of Temperature Stress on a Protein Library Across pH and Concentration using Microfluidic Modulation Spectroscopy

Abstract

Thermal stress is themost common stress condition utilized for gauging protein stability. However, techniques to measure the effects of thermal stress such as UV melts, circular dichroism(CD) and differential scanning calorimetry (DSC) typical only provide an overall melt temperature, Tm, and thermodynamic parameters, with very low granularity on structural changes associated with the melt. Microfluidic Modulation Spectroscopy (MMS) is an automated mid-IR technique capable of measuring protein secondary structure at very high resolution across a broad range of concentrations and buffer conditions. MMS can be used to measure and monitor the structural changes that are occurring, leading to protein unfolding, and then thermally induced aggregation. In this study, we started by measuring thermal unfolding for lysozyme across a range of pH values and found the Tm to be pH dependent. We next melted an IgG across a range of concentrations and found the Tm to be modestly concentration independent. Finally, we used MMS to determine the structural differences of the two Tm values determined for ovalbumin.