Biophysical Characterization of TRBP and Pdx1

Open Access
Author:
Evans, Declan Marshall
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Scott Showalter, Thesis Supervisor
  • Wendy Hanna-Rose, Honors Advisor
Keywords:
  • RNA
  • IDP
  • TRBP
  • Pdx1
Abstract:
Proteins are a diverse class of biomolecule with extreme importance to life. As the number of discovered proteins increases so too does the diversity of this class of molecule. Grand dogma that once governed our understanding of all proteins are being narrowed into specific classes. As a result old methods of studying proteins are becoming obsolete and new ones must be refined. This study focuses on two frontiers of protein chemistry; protein- RNA interactions and intrinsically disordered proteins. The discovery of catalytic RNA in the 1980’s forever changed understanding of enzymes. The focus of enzymology shifted from exclusive study of proteins to include RNA and protein- RNA complexes. RNA is now known to play key roles in many different regulatory pathways. One widely studied example is RNA interference (RNAi), a type of post- transcriptional gene regula- tion. RNAi involves the use of a protein- RNA complex called the RNA induced silencing complex (RISC). The RISC is composed of microRNA (miRNA) and several proteins including Dicer and TRBP. While there is extensive data as to the catalytic nature of the RISC, little is known about the protein-RNA interface. This study examines the HIV-1 TAR RNA binding protein (TRBP) and the thermodynamics associated with its RNA binding event. Isothermal titration calorimetry (ITC) is used to determine this binding event is not coupled with any large- scaled distortion in the A formed geometry of the RNA. Classical understanding of proteins has been rooted in our understanding of structure. Earliest crystalized proteins consisted of well formed domains of α-helices or β-sheets, and as a result, most analytic techniques are designed to probe these properties of proteins. Recently however, an i increasing number of proteins have been discovered that possess little to no secondary structure. Conventional methods have little success analyzing these proteins and novel methods must be created and refined. Here we examine the pancreatic and duodenal homeobox 1 (Pdx1), a member of the homeodomain family of transcription factors lacking large scale secondary structure outside of its DNA binding homeodomain. This protein is of particular interest due to its ties to diabetes. In this study we investigate the structure of Pdx1 and how changes can be linked to disease.