Abstract
Molecular chaperones play an essential role in protein quality control by stabilizing the non-native state of protein substrates, “clients,” to facilitate refolding, maturation, or degradation. Clients often interact with a coordinated system of chaperones; however, the molecular mechanisms of such interactions are not well-understood. An example is the transfer of clients between Hsp40/Hsp70 chaperones, in which Hsp40 promotes ATP hydrolysis by Hsp70 to allow for client transfer from Hsp40 to Hsp70. My thesis focuses on Hsp40/Hsp70 family members specific to the endoplasmic reticulum: ERdj3 (an Hsp40), and BiP (an Hsp70). More specifically, I am investigating and how BiP ATP hydrolysis promoted by the conserved J-domain (JD) of ERdj3 can facilitate client transfer to BiP.Fluorescence polarization (FP) measurements show that the JD can bind to the “ATP conformation” of BiP but not the “ADP-conformation” of BiP. ATPase measurements show that JD stimulates ATP hydrolysis by BiP. Förster resonance energy transfer (FRET) measurements indicate that the binding of JD to BiP and subsequent ATP hydrolysis causes BiP to significantly populate a conformational intermediate. I develop a quantitative model explaining the influence of JD on BiP, which suggests a conceptual mechanism by which client transfer from ERdj3 to BiP occurs.