Title: Investigation of PIP2-induced conformational movement within TMEM16A channels
Abstract:
TMEM16A is a chloride channel expressed on vascular endothelial and smooth muscle cells where it plays an important role in regulating blood pressure. TMEM16A channels require calcium and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to open. How PIP2 regulates TMEM16A channels is poorly understood but could be the foundation for targeting this channel in new treatments for cardiovascular diseases. I seek to understand how PIP2 binding changes TMEM16A channel conformation. Multiple structures of TMEM16A have been published; however, no structure has been resolved with an open pore or with PIP2 bound. Molecular dynamics simulations predict that transmembrane domains 4 and 6 move with PIP2 binding to enable chloride conductance. I will test this model using transition metal Forster Resonance Energy Transfer (tmFRET), which is a photon-less transfer of energy between a fluorescent donor and a nonfluorescent transition metal acceptor. I will take advantage of the ability of transition metals Ni2+ and Co2+ to bind to the Ca2+ binding pocket and activate the channel, which will enable me to use these metals as acceptor molecules. For these experiments, I will incorporate a fluorescent acceptor amino acid, Anap, at various transmembrane domain sites and measure changes in tmFRET efficiency. By depleting PIP2, I will uncover lipid-induced movement by measuring the changes in distance between the donor and acceptor sites.
Title: Phylogenetic Analysis of Transcription Elongation Factors
Abstract:
In eukaryotes, transcription of protein-coding genes is carried out by the DNA-dependent RNA Polymerase II (RNAPII) enzyme. Transcription elongation factors (TEFs) associate with RNAPII during transcription elongation and regulate the elongation rate and processivity of RNAPII and/or interface with nucleosomes to enable proper transcription of chromatinized DNA templates. However, since TEFs have only been studied in a few model organisms, the extent of conservation of TEFs and their functions across eukaryotes remains relatively unexplored. It is also unknown if TEFs in other species perform previously unrecognized functions. We have undertaken a phylogenetic approach to gain insights into the functions and evolutionary conservation of TEFs. We performed a Hidden-Markov Model (HMM) based search to identify putative homologs of TEFs in 304 species across the Tree of Life. To assess the functional conservation and variation of TEFs across the Tree of Life, we are utilizing the putative homolog sequences to identify conserved residues and clade-specific sequence features and domain architectures. Additionally, we are performing evolutionary rate co-variation (ERC) analyses to identify potential functional interactions with other proteins that might coordinate with TEFs during transcription. Such analyses will help guide future experiments exploring the functional landscape of TEFs during transcription. Furthermore, this study will provide insights into aspects of transcriptional regulation that are conserved across the Tree of Life.
Carlson and Arndt Lab
Friday, October 4th, 2024
12:00PM
Langley A219B