Detecting and directing molecular interactions that switch the functional state of G-protein coupled receptors
Final Report Abstract
The applicability of SMFS to characterize membrane proteins in vitro is developing rapidly and opening a wide range of fascinating possibilities to study how intra- and intermolecular interactions determine their structural stability and functional state. In particular, understanding how molecular interactions contribute to the functional state of GPCRs is of importance because they mediate most of our physiological responses and act as therapeutic targets for a broad spectrum of diseases. In our project we focused on SMFS to characterize GPCRs embedded in their physiologically relevant membranes and exposed to physiologically relevant conditions. SMFS uses a molecularly sharp stylus to grasp the terminal end of a GPCR and to quickly unfold the receptor while recording interaction forces. The positional accuracy of SMFS localizes these interactions to structural segments of the GPCR whereas the sensitivity of SMFS enables their stabilizing interaction forces to be quantified. To further investigate the kinetic, energetic and mechanical properties of the structural segments, dynamic SMFS (DFS) probes their stability over a wide range of loading rates. These parameters provided unique insight into the energy landscape that provides information on the structural and functional properties of the GPCRs. The selected highlights exemplify the application of SMFS to characterize inter- and intramolecular interactions, which change the properties of GPCRs in relation to their functional state (e.g., ligand binding), diseased state (e.g., mutation), or lipid environment such as cholesterol.
Publications
- ‘Modulation of molecular interactions and function by rhodopsin palmitylation’. Biochemistry (2009) 48, 4294-4304
P.S.H. Park, K.T. Sapra, B. Jastrzebska, T. Maeda, A. Maeda, W. Pulawski, M. Kono, J. Lem, R.K. Crouch, S. Filipek, D. J. Müller & K. Palczewski
- ‘Conservation of molecular interactions stabilizing bovine and mouse rhodopsin’. Biochemistry (2010) 49, 10412-10420
S. Kawamura, A.T. Colozo, D.J. Muller & P.S.H. Park
- ‘Cholesterol increases kinetic, energetic and mechanical stability of the human β2 adrenergic receptor’. Proc. Natl. Acad. Sci. USA (2012) 109, E3463-3473
M. Zocher, C. Zhang, G.F.S. Rassmussen, B.K. Kobilka & D.J. Muller
- ‘Ligand-specific interactions modulate kinetic, energetic and mechanical properties of the human β2 adrenergic receptor’. Structure (2012) 20, 1391-1402
M. Zocher, J.J. Fung, B.K. Kobilka & D.J. Muller
- ‘Structural, energetic and mechanical perturbations in a rhodopsin mutant that causes congenital stationary night blindness’. Journal of Biological Chemistry (2012) 287, 21826-21835
S. Kawamura, A.L. Colozo, L. Ge, D.J. Muller & P.S.H. Park
- ‘Single-molecule force spectroscopy of G-protein coupled receptors’. Chemical Society Reviews (2013) 42, 7801-7815
M. Zocher, Ch. Bippes, C. Zhang & D.J. Muller
- ‘Structural, kinetic, and mechanical differences between dark-state rhodopsin and opsin’. Structure (2013) 21, 426-437
S. Kawamura, M. Gerstung, L. Colozo, J. Helenius, N. Beerenwinkel, P.S.H. Park & D.J. Muller
