Yeast ribosome processing and assembly is a highly regulated and dynamic process that involves over 100 accessory proteins.  To date, fifteen DEAD-box proteins (unwindases) are implicated in modulating RNA structure and RNA-protein interactions.  DEAD-box proteins are enzymes that utilize ATP binding and hydrolysis to assist in the rearrangement of various RNA-protein complexes.  These proteins typically undergo a conformational change upon binding both ATP and RNA that substantially enhances ATP hydrolysis activity. Specifically, improvement of ATPase activity is mediated by the opening and closing of the ATP binding site.  We have improved thermal-fluorescence and limited-proteolysis assays to consistently monitor the presences of various conformational states in proteins that form ternary complexes involving ATP.  For example, our initial model enzyme, Hexokinase, undergoes various conformational changes upon glucose and ATP binding to form an activated 'closed' state.  Limited-proteolysis and tryptophan fluorescence experiments suggest that each ligand (ATP and glucose) induces distinct structural changes with varying stabilities.  Our thermal analysis reveals a two step unfolding transition in Hexokinase.   Interestingly, the binding of glucose or ATP stabilizes particular areas within the Hexokinase structure that results in fluorescent-thermal shifts since the hydrolysis rate of Rok1p, a yeast ribosomal DEAD-box protein, is not enhanced by RNA.  This observation suggests that Rok1p can initially adopt a closed-active conformation in the absence of RNA. To understand the initial conformational state of DEAD-box proteins, we have adapted our thermal-shift assays to monitor the structural stability of the yeast DEAD-box protein Rok1p.

Structural analysis of various conformational states in ATP binding proteins

247th National Meeting (2014)

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