One of the most challenging tasks remaining in the field of biochemistry is the one of understanding how the information within the amino acid sequence of proteins translates into a unique structure. Solving this problem would lead to endless possibilities for application in the medical and biotechnology industry. Many decades ago scientists realized that the process that facilitates the folding of a polypeptide chain could not be random and happen by chance; there needs to be direction in the folding free energy landscape. This landscape is defined by the thermodynamic factors entropy and enthalpy. The contribution made by enthalpy i.e. the contact energies from intra- and intermolecular interactions have been extensively investigated by various mutational studies. The influence of entropy on the other hand, is less well understood. My work focuses on the effect of altering the entropic components of forming the various parts of a known protein scaffold. This is done by genetic engineering in combination with biophysical characterisation and analysis. The results show effects on protein folding rates as well as on the pathway for nucleation and emphasis the ability of the folding landscape to readjust to entropic variations. Proteins are therefore not required to fold along a unique route to their final structure but can do so in several ways. The folding pathways we observe today have hence likely evolved as an adaptation to biological demands.

<p>Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found. </p><p>This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.</p>

Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found. This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.

<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