Rhodopsin Simulations in Detergent Micelles Characterize Meta I and Meta II Photointermediates

Abstract

G protein-coupled receptors (GPCRs) are prominent pharmaceutical targets due to their prevalence in the human genome. Membrane proteins such as GPCRs have been historically difficult to characterize by crystallographic methods, severely limiting development of therapeutical applications. A commonly used in vitro method for studying GPCRs is detergent solubilization, which can retain protein activity but affects thermodynamics and conformational flexibility. We used molecular dynamics (MD) to simulate the canonical GPCR rhodopsin in micelles containing the detergent DDM or CHAPS. The Meta I ↔ Meta II activation mechanism of rhodopsin is forward-shifted in a DDM micellar system [1], whereas it is back-shifted when solubilized in CHAPS. Previous MD simulations of rhodopsin could not reach Meta II due to the long timescale required (ms); in addition crystal structures of the activated state have been lacking [2-4]. However, detergents such as DDM accelerate Meta II kinetics to the s timescale [1], making it possible to reach the Meta II state in a DDM micelle. Using the recently published crystal structure of a putative Meta II state [5], we are able to show how the forward- and back-shifting of the Meta I ↔ Meta II equilibrium takes place in DDM and CHAPS detergent environments, respectively. These results provide important structural insights into rhodopsin activation, which can be extended to other GPCRs in a membrane mimetic environment, such as the A2A adenosine receptor. We have introduced a powerful tool which allows us to examine crystallization effects and isolate GPCR activated states.[1] S. Jager et al. (1997) Biochemistry 36:1999.[2] K. Martinez-Mayorga et al. (2006) JACS 128:16502.[3] P.-W. Lau et al. (2007) JMB 372:906.[4] T. Huber et al. (2004) Biophys. J. 86:2078.[5] H.-W. Choe et al. (2011) Nature 471:651.