Constructing a Fluorescence-Based Technique to Model Protein Unfolding
Mentor 1
Ionel Popa
Start Date
28-4-2023 12:00 AM
Description
The unfolding and refolding of protein domains is a phenomenon which frequently occurs inside the human body. Protein misfolding is related to the formation of aggregates, which is a benchmark for debilitating conditions such as Alzheimer’s and Parkinson’s disease. In this study, we seek to construct a fluorescence-based technique to model protein unfolding under force, establishing a framework for discovery of mechano-active drugs that target protein misfolding. Our technique is based on the use of fluorescent environment-sensitive dyes, whose fluorescence intensity or wavelength changes with a change in environment. In the case of the fluorescent environment-sensitive dyes SYPRO Orange and 1-anilinonaphthalene-8-sulfonate (ANS), which we focus on in this study, the fluorescence intensity of the dye molecules increases when they bind to hydrophobic regions within Bovine Serum Albumin (BSA) proteins. When BSA protein domains unfold, dye molecules are released from these pockets, causing an overall decrease in fluorescence that indicates a change in folded state. We induce protein unfolding and measure the resulting change in fluorescence using our Force Clamp (FC) hydrogel rheometry approach, in which a controlled pulling force unfolds and extends the domains of crosslinked proteins within protein hydrogels, which are elastic protein-based biomaterials that simulate tissue. Our aim is to quantify the number of unfolded domains in a protein as a function of mechanical stress on the domains. As chemical denaturants and mechanical forces both cause denaturation in proteins, measuring the effect of chemical denaturation allows us to quantify the number of unfolded domains in BSA as a function of protein denaturation and thus as a function of mechanical stress. Using a fluorescence marker for unfolding will allow us to analyze the viscoelastic response of protein-based materials and separate network elasticity due to molecular rearrangements from mechanical unfolding.
Constructing a Fluorescence-Based Technique to Model Protein Unfolding
The unfolding and refolding of protein domains is a phenomenon which frequently occurs inside the human body. Protein misfolding is related to the formation of aggregates, which is a benchmark for debilitating conditions such as Alzheimer’s and Parkinson’s disease. In this study, we seek to construct a fluorescence-based technique to model protein unfolding under force, establishing a framework for discovery of mechano-active drugs that target protein misfolding. Our technique is based on the use of fluorescent environment-sensitive dyes, whose fluorescence intensity or wavelength changes with a change in environment. In the case of the fluorescent environment-sensitive dyes SYPRO Orange and 1-anilinonaphthalene-8-sulfonate (ANS), which we focus on in this study, the fluorescence intensity of the dye molecules increases when they bind to hydrophobic regions within Bovine Serum Albumin (BSA) proteins. When BSA protein domains unfold, dye molecules are released from these pockets, causing an overall decrease in fluorescence that indicates a change in folded state. We induce protein unfolding and measure the resulting change in fluorescence using our Force Clamp (FC) hydrogel rheometry approach, in which a controlled pulling force unfolds and extends the domains of crosslinked proteins within protein hydrogels, which are elastic protein-based biomaterials that simulate tissue. Our aim is to quantify the number of unfolded domains in a protein as a function of mechanical stress on the domains. As chemical denaturants and mechanical forces both cause denaturation in proteins, measuring the effect of chemical denaturation allows us to quantify the number of unfolded domains in BSA as a function of protein denaturation and thus as a function of mechanical stress. Using a fluorescence marker for unfolding will allow us to analyze the viscoelastic response of protein-based materials and separate network elasticity due to molecular rearrangements from mechanical unfolding.
