<p>Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics. </p><p>During the resent decade, the importance of a certain residue in structure formation has been deduced from Φ-value analysis. As a complement to Φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, Φ´(β^TS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest Φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to overcome the entropic barrier. </p><p>Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β-α-β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from<i> A. aeolicus</i> even though the nucleation and core composition of this protein differ from other related structure-homologues. </p><p>In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electrostatic effects. By pH titration and protein engineering the charge content of S6 from <i>T. thermophilus</i> was altered and the results show that the charged groups at the protein surface might not be crucial for protein stability but, indeed, have impact on folding kinetics. Furthermore, by site-specific removal of all acidic groups the entire pH dependence of protein stability was depleted.</p>

Proteins are among the most complex molecules in the cell and they play a major role in life itself. The complexity is not restricted to just structure and function, but also embraces the protein folding reaction. Within the field of protein folding, the focus of this thesis is on the features of the folding transition state in terms of growing contacts, common nucleation motifs and the contribution of charged residues to stability and folding kinetics. During the resent decade, the importance of a certain residue in structure formation has been deduced from Φ-value analysis. As a complement to Φ-value analysis, I present how scatter in a Hammond plot is related to site-specific information of contact formation, Φ´(βTS), and this new formalism was experimentally tested on the protein L23. The results show that the contacts with highest Φ growth at the barrier top were distributed like a second layer outside the folding nucleus. This contact layer is the critical interactions needed to be formed to overcome the entropic barrier. Furthermore, the nature of the folding nucleus has been shown to be very similar among proteins with homologous structures and, in the split β-α-β family the proteins favour a two-strand-helix motif. Here I show that the two-strand-helix motif is also present in the ribosomal protein S6 from A. aeolicus even though the nucleation and core composition of this protein differ from other related structure-homologues. In contrast to nucleation and contact growth, which are events driven by the hydrophobic effect, my most recent work is focused on electrostatic effects. By pH titration and protein engineering the charge content of S6 from T. thermophilus was altered and the results show that the charged groups at the protein surface might not be crucial for protein stability but, indeed, have impact on folding kinetics. Furthermore, by site-specific removal of all acidic groups the entire pH dependence of protein stability was depleted.

To investigate how protein folding is affected by sequence connectivity five topological variants of the ribosomal protein S6 were constructed through circular permutation.  In these constructs, the chain connectivity (i.e. the order of secondary-structure elements) is changed without changing the native-state topology.  The effects of the permutations on the folding process were then characterised by φ-value analysis, which estimates the extent of contact formations in the transition-state ensemble.  The results show that the folding nuclei of the wild-type and permutant proteins comprises a common motif of one α-helix docking against two β-sheets, i.e. the minimal structure for folding.  However, this motif is recruited in different parts of the S6 structure depending on the permutation, either in the α1 or α2 half of the protein.  This minimal structure is not unique for S6 but can also be seen in other proteins.  As an effect of the dual nucleation possibilities, the transition-state changes describe a competition between two parallel pathways, which both include the central β-stand 1.  This strand constitutes thus a structural overlap between the two competing nuclei.  As similar overlap between competing nuclei is also seen in other proteins, I hypothesise that the coupling of several small nuclei into extended ‘super nuclei’ represents a general principle for propagating folding cooperativity across large structural distances.  Moreover, I demonstrate by NMR analysis that the existence of multiple folding nuclei renders the H/D-exchange kinetics independent of the folding pathway. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper IV: Manuscript