Structure Based Protein Models and In Vitro Experiments to Investigate the Folding Landscape of the Human Obesity Protein Leptin

Abstract

Jeffery Friedman discovered the human obesity gen in 1994. The expressed hormone leptin plays a key role in regulating energy intake/expenditure through the JAK/STAT signaling pathway. Even though, biologically, it is a well-studied protein not much is known about the biophysical properties.The mature protein is 146 residues (16kDa) buildup of 5 α-helixes and one stabilizing disulfide bond. This disulfide bridge encloses the C-terminal cysteine with residue 96 into a 50 residues long loop, creating a ‘so-called’ cysteine knot.In this research we explore the free energy landscape through structure-based protein models (Cα- and all-atom) and experiments (CD and fluorescence). The result shows that leptin can fold both in its reduced- and oxidized state. Even though the oxidized protein has a non-trivial pathway, where the N-terminal has to ‘thread’ through the loop to reach the native state, the folding landscape looks almost the same as for the reduced protein. The first event of folding is to form the loop or horseshoe (in the reduced state) constructed of α4 and α5 to stabilize the structure. Secondly, α3 is pulled into position: Either by a slipknot, backing into the loop in the oxidized state or by being pulled back to the correct conformation behind the horseshoe in the reduced state. The dragging force for this slipknot event is pulled from the energetic gain of zipping α2 together with α5. The last step is to fold α1 into its native conformation in the back of the structure between α5 and α2.