The protein folding problem is one of the most important issues to be solved in the field of molecular biology. The subject of this thesis mainly deals with various aspects of the folding process.

In Paper I, near-UV CD kinetic measurements on mutants, in which one tryptophan (Trp) residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCA II). The development of the individual (Trp) CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCA II at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Three Trp residues were analyzed, each probing different structural regions of the native structure. The investigated Trp residues develop their native CD bands at different rates, showing that formation of native-like tertiary structure is occurring with varying rates indifferent regions of the protein.

The same approach was applied to the extracellular domain of human tissue factor (sTF), which contains four Trp residues (Paper II). The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated, including the far-UV region. This leads to uncertain predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp-free CD spectrum. The kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.

In Paper III, the role of the metal ion cofactor for the refolding of bovine carbonic anhydrase II (BCA II) was studied from the molten globule to the native state. Refolding was possible to achieve by mere addition of the metal ion to the apomolten-globule, because the apoenzyme was less stable than the holoenzyme. The cofactor-effected refolding can be summarized as follows: 1) initially, the metal ion binds to the molten globule; 2) compaction of the metal-binding site region is then induced by the metal ion binding; 3) a functioning active center is formed; 4) finally, the native tertiary structure is generated in the outer parts of the protein.

In paper IV the aim was to determine the nature of the tetramer contact of human extracellular superoxide dismutase (hEC-SOD). We chose a strategy in which we mapped the subunit interaction interface by studying effects of twelve different mutations in the N-terminal domain fused to HCA II. The results show that the hydrophobic side of a predicted amphiphatic a-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.