A Comparative Study of Fluorescent Dyes for Protein Folded State Detection
Mentor 1
Ionel Popa
Start Date
10-5-2022 10:00 AM
Description
Protein hydrogels are elastic materials composed of folded domains that serve as useful experimental models for tissues and constitute a promising method to study mechanical unfolding of proteins in bulk. Our lab has developed a Force Clamp (FC) hydrogel rheometry approach to apply a controlled force to protein hydrogels, simultaneously unfolding and extending protein domains. Due to the random cross-linking geometry of domains inside hydrogels, which affects protein mechanical stability, a fluorescence-based method is highly desirable as a method of decoupling mechanical unfolding from the intrinsic elasticity of the network. Here we explore fluorescent environment-sensitive dyes, such as SYPRO Orange and 1-anilinonaphthalene-8-sulfonate (ANS), as useful indicators of a protein’s folded state inside protein hydrogels. When hydrogels are exposed to stress in the presence of either dye, dye molecules that bind to the hydrophobic regions of proteins inside the hydrogel exhibit an overall decrease in fluorescence intensity as proteins unfold. Our goal in this study is to determine, using the protein Bovine Serum Albumin (BSA), which dye is a better indicator of folded and unfolded states. More specifically, the dye that exhibits the greatest change in fluorescence when bound to BSA will be used in future experiments characterizing the unfolding mechanism and mechanical stability of BSA. Having a clear indicator of a change in a protein’s folded state will allow us to establish a framework for discovery of mechano-active drugs that target protein misfolding, allowing us to make strides in the fight against protein misfolding diseases such as Alzheimer’s Disease. Thus far, we have studied the fluorescent and binding behavior of ANS. Our results suggest a 3:1 binding ratio between ANS and BSA in both gel and aqueous form, which agrees with previous studies while challenging the results of other studies.
A Comparative Study of Fluorescent Dyes for Protein Folded State Detection
Protein hydrogels are elastic materials composed of folded domains that serve as useful experimental models for tissues and constitute a promising method to study mechanical unfolding of proteins in bulk. Our lab has developed a Force Clamp (FC) hydrogel rheometry approach to apply a controlled force to protein hydrogels, simultaneously unfolding and extending protein domains. Due to the random cross-linking geometry of domains inside hydrogels, which affects protein mechanical stability, a fluorescence-based method is highly desirable as a method of decoupling mechanical unfolding from the intrinsic elasticity of the network. Here we explore fluorescent environment-sensitive dyes, such as SYPRO Orange and 1-anilinonaphthalene-8-sulfonate (ANS), as useful indicators of a protein’s folded state inside protein hydrogels. When hydrogels are exposed to stress in the presence of either dye, dye molecules that bind to the hydrophobic regions of proteins inside the hydrogel exhibit an overall decrease in fluorescence intensity as proteins unfold. Our goal in this study is to determine, using the protein Bovine Serum Albumin (BSA), which dye is a better indicator of folded and unfolded states. More specifically, the dye that exhibits the greatest change in fluorescence when bound to BSA will be used in future experiments characterizing the unfolding mechanism and mechanical stability of BSA. Having a clear indicator of a change in a protein’s folded state will allow us to establish a framework for discovery of mechano-active drugs that target protein misfolding, allowing us to make strides in the fight against protein misfolding diseases such as Alzheimer’s Disease. Thus far, we have studied the fluorescent and binding behavior of ANS. Our results suggest a 3:1 binding ratio between ANS and BSA in both gel and aqueous form, which agrees with previous studies while challenging the results of other studies.
