Comparison of stability predictions and simulated unfolding of rhodopsin structures
Oznur Tastan, Esther Yu, Madhavi Ganapathiraju, Anes Aref, AJ Rader and Judith Klein-Seetharaman
Abstract:
Developing a better mechanistic understanding of membrane protein folding is urgently needed due to 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 3Å using the Floppy Inclusions and Rigid Substructure Topography (FIRST) method [Jacobs, D. J., Rader, A. J., Kuhn, L. A. & Thorpe, M. F. (2001) Proteins 44, 150-165] described previously for a single mammalian rhodopsin structure [Rader et al. (2004) PNAS 101, 7246-7251]. 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.
Supplementary Tables:
1) Supplementary Table 1: Experimental data compiled for BR and MR folding and stability changes. Please refer to the paper for the description of the data.
2) Supplementary Table 2: Protein structures used in the study.
Additional Supplementary
Files:
1) To check whether there is biasing of the results by the better resolved BR structures as compared to mammalian rhodopsin structures, we examined the subset of the BR structures where the resolution is comparable to that of the set of mammalian rhodopsin structures. You can find the results here.
2) By clicking on the links in Table 4 you will obtain access to directories that include FIRST simulated unfolding related data for the protein structures listed in Table 3.
Table3 : PDB ids of the 62 rhodopsin crystal structures with resolution greater than 3 A downloaded from the Protein Databank in June 2006.
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Mammalian Rhodopsin (5)* |
Sensory Rhodopsin II (4) |
Sensory Rhodopsin II
with Transducer (3) |
Anabaena
Sensory Rhodopsin (1) |
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1f88 1gzm 1hzx 1l9h 1u19 |
1gu8 1gue 1h68 1jgj |
1h2s 2f93 2f95 |
1xio |
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Halorhodopsin (1) |
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1e12 |
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Bacteriorhodopsin
(48) |
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1ap9 1brr 1brx 1c3w 1c8r 1c8s 1cwq 1dze 1e0p 1f4z 1f50 1iw6 |
1iw9 1ixf 1jgj 1jv6 1jv7 1kg8 1kg9 1kgb 1kme 1m0k 1m0l 1m0m |
1o0a 1p8h 1p8i 1p8u 1pxr 1pxs 1py6 1q5i 1q5j 1qhj 1qko 1qkp |
1qm8 1s51 1s52 1s53 1s54 1tn0 1tn5 1ucq 1x0i 1x0k 1x0s 1xji |
*The number in brackets indicates the number of structures for the corresponding rhodopsin.
Table 4: The FIRST simulated unfolding results for all 62 rhodopsin crystal structures (listed in the table above).
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Mammalian Rhodopsin |
Bacteriorhodopsins |
Halorhodopsin |
Anabaena Rhodopsin |
Sensory Rhodopsins crystallized without the transducer |
Sensory Rhodopsins crystallized with the transducer and analyzed in the presence of the transducer |
Sensory Rhodopsins crystallized with the transducer and analyzed in the absence of the transducer |
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Dilution Plots *ps and *pdf |
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Folding Core and FC Energies *fc.txt |
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Folding Cores on 3D structure1 |
1 Please download the *_RCD.pdb and *.pml file in the same folder and run the *.pml script with Pymol. In the manuscript the largest rigid cluster and the second largest cluster were depicted as red and blue to coincide with the dilution plots. Here the color coding is FIRST's default coding in which blue is the largest rigid cluster and red is the second largest rigid cluster.
Last updated June 2006.