Linköping Studies in Science and Technology. Dissertations
No. 533

The Interaction Between the Chaperonin GroEL and Human Carbonic Anhydrase II
Protein Folding and Stability Studies

Malin Persson

Akademisk avhandling

som för avläggande av filosofie doktorsavhandling vid Linköpings Universitet kommer att offentligt försvaras i hörsal Planck, Fysikhuset, Linköpings Universitet, fredagen den 15 maj 1998, kl. 10.15. Opponent är Prof. Gunnar von Heijne, Stockholms Universitet

ABSTRACT

The presence of GroEL/ES during the refolding of human carbonic anhydrase II was found to increase the yield of active enzyme from approx. 70 to approx. 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

The chaperonin GroEL is a heat shock protein, i.e. it protects other proteins when they are exposed to elevated temperatures. To investigate if GroEL protects HCA II from thermal denaturation we studied the interaction between GroEL and HCA II at different temperatures. The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35°C. At temperatures above 35°C, the enzymatic activity is not stable, but decreases over time. If GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50°C. However, unlike the spontaneous reactivation, the enzymatic activity is stable with time in the presence of GroEL. In the presence of the chaperonin, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present. It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme. Furthermore, proline isomerization is part of the rate limiting step of refolding even in the presence of GroEL, and it is noteworthy that prolyl isomerase will influence the refolding of HCA II in the presence of GroEL.

The kinetics of the refolding of HCA II, at different temperatures, together with GroEL, has also been studied. The Arrhenius plots for the spontaneous, GroEL-assisted, and GroEL/ES-assisted refolding of HCA II show that the apparent activation energy (Ea) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES-assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES-assisted reactions, respectively). This is the first indications that point to an active role for GroEL in the protein-folding process. Hence, GroEL does not only protect HCA II during refolding, it also assists the protein in the refolding reaction by providing a folding route with a flatter energy landscape than the spontaneous reaction.

To further investigate the interaction between GroEL and HCA II, we have used electron paramagnetic resonance (EPR) to study HCA II cysteine mutants, spin-labeled at specific positions of the protein molecule. From the change of the mobility at temperatures between 20°C and 50°C of the spin-label for various mutants, with and without the presence of GroEL, the following general observations were made; i) HCA II appears to be unfolded to a degree similar to that of a GuHCl-induced molten-globule intermediate of the enzyme for the initial interaction with GroEL; ii) the degree of binding to GroEL is dependent on the stability of the HCAII variant; iii) GroEL efficiently protects HCAII from irreversible aggregation at higher temperatures; iv) the GroEL interaction leads to higher flexibility of the rigid and compact hydrophobic core, which is likely to facilitate rearrangements of misfolded structure; v) protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements.

Keywords: carbonic anhydrase, protein folding, protein stability, chaperone, GroEL, folding mechanism, site-directed spin-labeling, electron paramagnetic resonance (EPR).

Department of Physics and Measurement Technology, Chemistry
Linköpings Universitet, S-581 83 Linköping, Sweden
Linköping 1998

ISBN 91-7219-206-2 ISSN 0345-7524