The ultimate goal of protein folding studies is to be able to predict folding pathways from amino acid sequence. This goal is still elusive but experiment and simulation are beginning to converge.
A new method to assess whether curved chevron plots are due to a switch between two barriers or to movement of the transition state:
Gianni, S., Brunori, M., Jemth, P., Oliveberg, M., and Zhang, M. (2009) Distinguishing between narrow and broad free energy barriers in protein folding. Biochemistry 48, 11825-11830 [abstract] We have performed folding studies on PDZ domains and found that they fold with a similar kinetic mechanism, involving a high-energy intermediate, as shown in the papers below.
The intermediate in the folding reaction of PDZ domains appears to be generic. We analyzed the folding reaction of five PDZ domains and all could be described by a model with two transition states and a high energy intermediate. Read more about it in:
Chi, C., Gianni, S.*, Calosci, N., Travaglini-Allocatelli, C., Engström, Å., and Jemth, P.* (2007) A conserved folding mechanism for PDZ domains. FEBS Lett. 581, 1109-1113 [abstract]
The intermediate in the folding reaction of a PDZ domain is on-pathway! We use ligand induced folding as a new approach to prove that the change of rate-limiting step in the folding reaction of PTP-BL PDZ2 is due to an on-pathway intermediate. Read more about it in:
Ivarsson, Y., Travaglini-Allocatelli, C., Jemth, P., Malatesta, F., Brunori, M., and Gianni, S.* (2007) An on-pathway intermediate in the folding of a PDZ domain. J. Biol. Chem. 282, 8568-8572 [abstract]
Furthermore, structural characterization of the two transition states nicely illustrates the folding energy landscape:
Calosci, N., Chi, C. N., Richter, B., Camilloni, C. Engström, Å, Eklund, L., Travaglini-Allocatelli, C., Gianni, S.*, Vendruscolo, M.* and Jemth, P.* (2008) A comparison of the successive transition states for folding reveals alternative early folding pathways. Proc. Natl. Acad. Sci. USA. 105, 19240-19245 [abstract]
Protein Binding and Allostery
We have also found evidence for a conformational change whithin a PDZ domain. The paper is about intradomain conformational changes in protein-ligand interactions and their possible functional role. By an ultra-rapid mixing device called continuous flow we were able to measure very high rate constants for the binding reaction of PDZ domain and peptide ligand. The kinetic data demonstrated that there is a conformational change in the PDZ domain upon binding. NMR data and mutational studies were also consistent with a conformational transition. Interestingly, the conformational change was only detected in one of the two PDZ domains that we studied, suggesting that different binding mechanisms may be exploited whithin the PDZ domain family.
Gianni, S., Walma, T., Arcovito, A., Calosci, N., Bellelli, A., Engström, Å., Travaglini-Allocatelli, C., Brunori, M., Jemth, P.*, and Vuister, G. W.* (2006) Demonstration of long-range interactions in a PDZ domain by NMR, kinetics and protein engineering. Structure 14, 1801-1809 [abstract]
We then explored yet another PDZ domain (SAP97/hDlg PDZ2) and found, similarly to PTP-BL PDZ2, that the conformational change observed by fluorescence spectroscopy follows an induced fit, or sequential, mechanism:
Chi, C. N., Bach, A., Engström, Å, Wang, H., Strømgaard, K., Gianni, S., and Jemth, P* (2009) A sequential binding mechanism in a PDZ domain. Biochemistry 48, 7089-7097 [abstract]
More about induced and selected fit: here the conformational change comes first!
Savino, C., Montemiglio, L.C., Sciara, G., Miele, A.E., Kendrew, S.G., Jemth, P., Gianni, S., and Vallone, B. (2009) Investigating the structural plasticity of a cytochrome P450: Three dimensional structures of P450 EryK and binding to its physiological substrate. J. Biol. Chem. In press [abstract]
Can sparse energetic networks in protein domains be predicted by multiple sequence analysis? We have experimentally tested the conserved network in PDZ domains and found that the residues involved do not communicate more than non-network residues. Read about it in:
Chi, C. N., Elfström, L., Shi, Y., Engström, Å, Snäll, T., and Jemth, P. (2008) Reassessing a sparse energetic network within a single protein domain. Proc. Natl. Acad. Sci. USA. 105, 4679-4684 [abstract]
Press release in Swedish
A current topics/perspectives review on the folding and binding reactions of PDZ domains is out! We present data supporting a challenging idea: that the residues important for the stability of a protein can be involved in its function. Read more about it in:
Jemth, P.* and Gianni, S.* (2007) PDZ domains: folding and binding. Biochemistry 46, 8701-8708 [abstract]
In my postoctoral work we studied the folding of an FF domain. The FF domain, a protein consisting of three alpha and one 3-10 helix, was found to fold via an on-pathway intermediate. The role (and even presence) of intermediates in folding reactions of protein domains is controversial. But, they seem to be there!
The folding mechanism of the FF domain was described in:
Jemth, P., Gianni, S., Day, R., Li, B., Johnson, C. M., Daggett, V., and Fersht, A. R. (2004) Demonstration of a low energy on-pathway intermediate in a fast folding protein by kinetics, protein engineering and simulation. Proc. Natl Acad. Sci. USA 101, 6450-6455 [abstract]
And cryptic intermediates are found here:
White, G., Gianni, S., Grossman, G., Jemth, P., Fersht, A. R., and Daggett, V. (2005) Simulation and experiment conspire to reveal cryptic intermediates and the slide from the nucleation-condensation to framework mechanism of folding. J. Mol. Biol. 350, 757-775 [abstract]
The major transition state for the folding reaction of the FF domain was studied by Phi-value analysis. This approach estimates the extent of native structure formation in the transition state by protein engineering, thermodynamics and kinetics. The results are described in:
Jemth, P., Day, R., Gianni, S., Khan, F., Allen, M. D., Daggett, V., and Fersht, A. R. (2005) The structure of the major transition state for folding of an FF domain from experiment and simulation. J. Mol. Biol. 350, 363-378 [abstract]
And finally, the intermediate in the folding reaction of the FF domain was detected by temperature-jump fluorimetry and shown to be present under various conditions:
Jemth, P., Johnson, C. M., Gianni, S., and Fersht, A. R (2008) Demonstration by burst phase analysis of a robust folding intermediate in the FF domain. Prot. Eng. Des. Sel. 21, 207-214 [abstract]
Here's the transition state for folding of the FF domain, grafted onto the native structure:
And here's the movie of the FF transition state for QuickTime Player (courtesy of Bob Robinson): FF TS movie [1 Mb]
Three reviews on protein folding:
Daggett, V., and Fersht, A. (2003) The present view on the mechanism of protein folding. Nat. Rev. Mol. Cell. Biol. 4, 497-502 [abstract]
Ferguson, N., and Fersht, A. R. (2003) Early events in protein folding. Curr. Opin. Struct. Biol. 13, 75-81 [abstract]
Travaglini-Allocatelli, C., Gianni, S., and Brunori, M. (2004) A common folding mechanism in the cytochrome c family. Trends Biochem. Sci. 29, 535-541 [abstract]
The structure of the FF domain:
Allen, M., Friedler, A., Schon, O., and Bycroft, M. (2002) The structure of an FF domain from human HYPA/FBP11. J. Mol. Biol. 323, 411-416 [abstract]