Protein folding and misfolding, chaperone function
Group leader: Uno Carlsson
During our long-going studies of protein folding we have developed new methods to monitor specific conformational changes that also can be applied to related important fields. Therefore, we have also proceeded to investigate protein misfolding and aggregation, protein-protein interaction, protein design as well as the mechanism of protein adsorption to solid surfaces, studies that can benefit from our experience in all aspects of protein folding.
It is fundamental for our understanding of the living cell to know the rules that direct the folding process and determine the final tertiary structure of a protein. Solving the protein folding problem is one of the most challenging tasks in the post-genomic era. Such knowledge would enable prediction of the 3-D structure and from that the biological function from the vast number of amino acid sequences of proteins now available.
Folding in vivo seems for many proteins to be assisted by several protein factors like molecular chaperones. Although these proteins can be folded spontaneously the chaperones suppress aggregation during folding and increase the yield. Insights into the mechanism of chaperone function are in themselves essential, but should in addition contribute to the understanding of the folding mechanism. Protein aggregation plays an important role in biotechnology and also causes numerous disorders, such as Alzheimer´s and prion diseases. Therefore, studies of the mechanism of misfolding and aggregation are also very important.
Several of theses aspects are addressed in our on-going research activities. The main model protein in these studies is human carbonic anhydrase II (HCA II). This protein is undoubtedly one of the best proteins now available for these kinds of studies, since it has the advantage to be monomeric, single-chain with no disulfide bonds, stable and intermediate in molecular weight. The structure has been well-defined biophysically and its structure-function relationship is very well characterized. Moreover, it forms detectable folding intermediates both at equilibrium and kinetically and is chaperoned by GroEL alone.
To be able to structurally characterize compact unfolded states in great detail that form during various stages of folding, we have pioneered the development of various types of site-directed Cys labeling techniques (fluorescent and spin labeling) that specifically monitor local changes in compactness, mobility and polarity during these processes (e.g. Svensson, 1995; Hammarström ,1997; 2001). This probing approach has also successfully been applied to studies of various forms of protein-protein interaction including protein aggregation (eg. Persson et al. 1999; Hammarström et al., 1999; Carlsson et al., 2006).
We have also been able to extend the use of our site-directed probing technique to real time measurements of specific domain docking during protein-protein association by combining stopped-flow fluorescence and SPR measurements. A new approach that takes into account and compensate for different global binding kinetics of the protein variants caused by mutagenesis and labelling (Österlund, et al., 2005b). To be able to resolve in detail how a protein is oriented when adsorbed to a solid surface we have combined site-directed fluorescent labeling and the use of nanoparticle sized surfaces that do not scatter light; a methodology that should be a powerful tool for specific mapping of the interaction area between an adsorbed protein and a solid surface (Karlsson et al. 2005).
We are now continuing our efforts to apply EPR measurements on doubly spin-labeled mutants for distance measurements at room temperature to be amenable for folding studies.
Characterization of folding intermediates
We have earlier demonstrated that HCA II undergoes a three-state unfolding process with formation of a molten globule intermediate (Carlsson et al.,2000). In a disulfide-bridge stabilized HCA II mutant this molten globule, which is prone to aggregation, was significantly suppressed (to 10 %) (Mårtensson et al, 2002). The reduction of the amount of molten globule during folding also led to significantly higher refolding yields compared to that of the wild-type (Karlsson et al., 2004).
Moreover, in the characterization of a H107Y mutant of HCA II, causing the disease marble brain syndrome, we have discovered a new form of molten globule, “molten globule light”, that exposes hydrophobic patches to a lesser extent than the classical molten globule and is therefore less prone to aggregate. Moreover, it is inactive and has a disrupted structure with maintained partial tertiary structure (Almstedt et al, 2004). To analyze this new molten globule state we have engineered several mutants that also have been shown to populate both the molten globule and the molten globule light state. The amount of molten globule states has been shown to be suppressed in all these variants and other mutants by a small molecule inhibitor, shifting the equilibrium towards the native state.
We are now testing a series of aromatic sulfonamide inhibitors to gain insight into how it would be possible to design a small molecule that prevents misfolding and might be used for therapeutical intervention.
To further investigate the devastating effect of the H107Y mutation, a mutational analysis (double mutant cycles) of this position and a neighboring interacting position E117 is under way, indicating that H107 and E117 are independently stabilizing the folded protein and that long-range effects are the cause for the noted H107Y destabilization.
Studies of chaperone function: GroEL and GroES mediated folding
One of the most interesting questions is how protection against misfolding and aggregation during the folding process is accomplished by chaperones.There has been a debate on whether the most studied chaperone GroEL not only protects against aggregation but also functions as an unfoldase by which activity correction of misfolded structure can occur.In a series of papers we have put forward strong evidences that GroEL unfolds its protein substrate during the interaction (Persson et al., 1999; Hammarström et al., 2000; 2001). However, in a recent review article additional evidences have been put forward supporting a more active role of GroEL (Lin and Rye, 2006). To estimate the magnitude of substrate protein unfolding by GroEL we have recently completed a study on a doubly spin-labeled HCA II for distance determinations. It was shown that that the labeled positions at a topological breakpoint in the protein β-sheet core are in proximity in the native state of HCA II (~8 Å), and that the local structure is virtually intact in the thermally-induced molten-globule state that binds to GroEL. Upon interaction with GroEL substantial increase in spin-spin distance (to ~20 Å) demonstrates a significant GroEL-induced conformational change in HCA II.
By using AEDANS-labeled HCA II variants we have by stopped-flow fluorescent measurements observed that GroEL stretches the protein substrate as an early event in the folding process, when compared to spontaneous folding. Interestingly, GroES alone can transiently interact with the folding protein leading to remodeling of the structure of the molten globule intermediate. Thus, GroES alone is able to induce stretching in the central part of the protein substrate and compression in the C-terminal region.
Chaperone function of proline isomerase (PPI or Cyp18)
Several years ago we put forward evidences for the first time that a PPI also can exhibit chaperone activity (Freskgård et al., 1992). This conclusion has been controversial due to a later detected slow kinetic refolding phase that might, at least in part, explain the higher refolding yields observed by us in the presence of PPI (Kern et al., 1994). We have now repeated our experiments on HCA II mutants that are much more prone to misfolding than the wild-type enzyme and shown that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e. function as a folding catalyst and a chaperone.
- Almstedt, K., M. Lundqvist, J. Carlsson, M. Karlsson, B. Persson, B.-H. Jonsson, , U. Carlsson, P. Hammarström (2004) J. Mol. Biol. 342, 619-633.
- Carlsson, U. & B.-H. Jonsson: (2000): Folding and stability of human carbonic anhydrase II. In The Carbonic Anhydrases. New Horizons. Birkhäuser, pp.241-262.
- Carlsson,K.,E.Persson, U.Carlsson, M.Svensson (2006) Biochem. Biophys. Res. Commun. 349, 1111-1116.
- Freskgård, P.O., N. Bergenhem, B.H. Jonsson, M. Svensson and U. Carlsson (1992) Science 258, 466-468.
- Hammarström, P., B. Kalman, B.-H. Jonsson, U. Carlsson (1997) FEBS Lett. 420, 63-68.
- Hammarström, P., M. Persson, P.-O. Freskgård, L.-G. Mårtensson, D. Andersson, B.-H. Jonsson, U. ---Carlsson (1999) J. Biol. Chem. 274, 32899-32903.
- Hammarström, P., M. Persson, R. Owenius, M. Lindgren,U. Carlsson (2000) J. Biol. Chem. 275, 22832-22838.
- Hammarström, P., M. Persson, U. Carlsson (2001) J. Biol. Chem. 276, 21765-21775.
- Karlsson, M., L.-G. Mårtensson, P. Olofsson & U. Carlsson (2004) Biochemistry 43, 6803-6807.
- Karlsson, M., U. Carlsson (2005) Biophys. J. 88, 3536-3544.
- Kern, G., Kern, D., Schmid, F.X. & Fischer, G. (1994) FEBS Lett. 348, 145-148.
- Persson, M., M. Lindgren, P. Hammarström, M. Svensson, B.-H. Jonsson, U. Carlsson (1999) Biochemistry 38, 432-441.
- Lin, Z., H.S. Rye (2006) Crit. Rev. Biochem. Mol. Biol. 41, 211-239.
- Mårtensson, L.-G., M. Karlsson, U. Carlsson (2002) Biochemistry 41, 15867-15875.
- Svensson, M, P.-O. Freskgård, M. Lindgren, K. Borén, U. Carlsson (1995) Biochemistry 34, 8606-8620.
- Österlund,M., E. Persson, U. Carlsson, P.-O. Freskgård, M. Svensson (2005) Biochem. Biophys. Res. Commun. 327, 1276-1282.
Responsible for this page: Maria Sunnerhagen
Last updated: 05/28/08