Leah Epstein, BA, MPhil
CUNY - Advanced Science Research Center
Functional Relevance of Construct Length on Protein Activity in the Blue-Light Sensing AsLOV2 Photoswitch
Phototropins are a family of blue-light activated kinases that control various cellular processes such as phototropism or stomatal opening in plants. One of the two LOV domains from oat Avena sativa phototropin 1, AsLOV2, has been used as a model system to understand the structural dynamics of signal transduction. Additionally, the simplicity of the AsLOV2 molecular switch has made it well-suited as the basis of numerous optogenetic tools. To gain further insight into this system, I used a combination of biochemical and structural techniques to understand the consequences of construct length on the degree of protein activation. High pressure NMR was used on two AsLOV2 constructs differing in the number of residues after the Jα helix, a major driving force in protein activation. We found slight differences in the chemical shift perturbations in the nonlinear pressure coefficient for three backbone nuclei (H, N, and CO) localized to the C-terminal end of AsLOV2 indicative of a greater degree of instability in the Jα region. Also we performed CPMG experiments on both constructs to understand how protein length contributes to baseline energetics available for engineering purposes. Finally, we validated the AsLOV2 signaling model within a larger LOV2-kinase construct using a combination of cryo-EM and HDX-MS, letting us understand the molecular mechanism underpinning the various conformational rearrangements the protein undergoes upon light activation within its native context.
Phototropins are a family of blue-light activated kinases that control various cellular processes such as phototropism or stomatal opening in plants. One of the two LOV domains from oat Avena sativa phototropin 1, AsLOV2, has been used as a model system to understand the structural dynamics of signal transduction. Additionally, the simplicity of the AsLOV2 molecular switch has made it well-suited as the basis of numerous optogenetic tools. To gain further insight into this system, I used a combination of biochemical and structural techniques to understand the consequences of construct length on the degree of protein activation. High pressure NMR was used on two AsLOV2 constructs differing in the number of residues after the Jα helix, a major driving force in protein activation. We found slight differences in the chemical shift perturbations in the nonlinear pressure coefficient for three backbone nuclei (H, N, and CO) localized to the C-terminal end of AsLOV2 indicative of a greater degree of instability in the Jα region. Also we performed CPMG experiments on both constructs to understand how protein length contributes to baseline energetics available for engineering purposes. Finally, we validated the AsLOV2 signaling model within a larger LOV2-kinase construct using a combination of cryo-EM and HDX-MS, letting us understand the molecular mechanism underpinning the various conformational rearrangements the protein undergoes upon light activation within its native context.
