Comparison of Stability Predictions and Simulated Unfolding of Rhodopsin Structures

Authors


  • This paper is part of the Proceedings of the 12th International Conference on Retinal Proteins held at Awaji Island, Hyogo, Japan on 4–8 June 2006.

*Corresponding author email: judithks@cs.cmu.edu (Judith Klein-Seetharaman)

Abstract

Developing a better mechanistic understanding of membrane protein folding is urgently needed because of the discovery of an increasing number of human diseases, where membrane protein instability and misfolding is involved. Towards this goal, we investigated folding and stability of 7-transmembrane (TM) helical bundles by computational methods. We compared the results of three different algorithms for predicting changes in stability of proteins against an experimental mutation dataset obtained for bacteriorhodopsin (BR) and mammalian rhodopsin and find that 61.6% and 70.6% of the mutation results can potentially be explained by known local contributors to the stability of the folded state of BR and mammalian rhodopsin, respectively. To obtain further information on the predicted folding pathway of 7-TM proteins, we conducted simulated thermal unfolding experiments of all available rhodopsin structures with resolution better than 3 Å using the Floppy Inclusions and Rigid Substructure Topography (FIRST) method (Jacobs, D. J., A. J. Rader, L. A. Kuhn and M. F. Thorpe [2001] Proteins44, 150) described previously for a single mammalian rhodopsin structure (Rader et al. [2004] PNAS101, 7246). In statistical comparison we found that structures of mammalian rhodopsin have a stability core that is characterized by long-range interactions involving amino acids close in space but distant in sequence comprising positions from both extracellular loop and TM regions. In contrast, BR-simulated unfolding does not reveal such a core but is dominated by interactions within individual and groups of TM helices, consistent with the two-stage hypothesis of membrane protein folding. Similar results were obtained for halo- and sensory rhodopsins as for BRs. However, the average folding core energies of sensory rhodopsins were in between those observed for mammalian rhodopsins and BRs hinting at a possible evolution of these structures toward a rhodopsin-like behavior. These results support the conclusion that although the two-stage model can explain the mechanisms of folding and stability of BR, it fails to account for the folding and stability of mammalian rhodopsin, even though the two proteins are structurally related.

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