Folding and aggregation studies in the acylphosphatase-like family / / Francesco Bemporad.

• Superior document: Premio FUP. Tesi di dottorato ; 6
• VerfasserIn: Bemporad, Francesco,
• Place / Publishing House: Firenze : : Firenze University Press,, 2009
• Year of Publication: 2009
• Language: English
• Series: Premio FUP. Tesi di dottorato ; 6.
• Physical Description: 1 online resource (126 pages) :; illustrations; digital, PDF file(s).

Folding and misfolding of proteins are considered two sides of the same coin. The delicate equilibrium existing between these two processes is crucial for any living organism and its alterations can lead to the onset of several tremendous diseases, such as Alzheimer's and Parkinson's disease. The attainment of a profound knowledge of folding/misfolding processes is a key step to understand how life works and for discovering new therapies to these diseases. In this work the author shows that proteins can display enzymatic activity even in the absence of a compact three-dimensional structure, with important implications for the study of protein enzymes. Furthermore, the author investigates the formation of protein aggregates similar to those observed in patients of amyloid-related diseases.

Table of Contents

Chapter -- Introduction -- 1.1 Different conformational states are populated during life-span of proteins -- 1.2 Protein folding -- 1.2.1 Definition of the protein folding problem -- 1.2.2 The early studies and the characterisation of intermediates -- 1.2.3 F value analysis and folding mechanisms: is there a unifying mechanism? -- 1.2.4 The role of topology in determining folding; the importance of studying structurally related proteins -- 1.3 Protein misfolding -- 1.3.1 Definition and structure of amyloid fibrils -- 1.3.2 Mechanisms of amyloid aggregation -- 1.4 Aim of this thesis -- 1.4.1 The acylphosphatase-like family -- 1.4.2 Folding and aggregation in the acylphosphatase-like family -- Chapter 2 Enzymatic activity in non-native Sso AcP -- 2.1 Introduction -- 2.2 Results -- 2.2.1 The partially folded state populated during folding of Sso AcP shows acylphosphatase activity -- 2.2.2 The acylphosphatase activity observed in the Sso AcP partially folded state is highly sensitive to mutations -- 2.2.3 Investigation of the partially folded and transition states of Sso AcP using F value analysis -- 2.3 Discussion -- 2.3.1 Structure of the partially folded state and of the transition state -- 2.3.2 Enzymatic activity in the presence of a highly dynamic catalytic site -- 2.3.3 Biological function in the absence of a three-dimensional fold -- 2.4 Materials and methods -- 2.4.1 Mutagenesis -- 2.4.2 Protein expression and purification -- 2.4.3 Enzymatic activity essay -- 2.4.4 Development of enzymatic activity during folding -- VIII Francesco Bemporad -- 2.4.5 Equilibrium GdnHCl-induced unfolding curves -- 2.4.6 Folding kinetics -- 2.4.7 Unfolding kinetics -- 2.4.8 F value analysis -- Chapter 3 Aggregation studies on Sso AcP -- 3.1 Introduction -- 3.1.1 Aggregation from native states -- 3.1.2 Aggregation of Sso AcP -- 3.2 Results -- 3.2.1 Sso AcP aggregates regardless of the position of the N-terminal segment -- 3.2.2 N-terminal segment does not induce Sso AcP aggregation via adestabilising effect -- 3.2.3 A specific inter-molecular interaction between N-terminal segment and globular Sso AcP leads to the formation of early aggregates -- 3.2.4 Formation of initial aggregates depends on protein concentration -- 3.2.5 Experiments with acrylodan -- 3.3 Conclusions: a possible aggregation mechanism for Sso AcP -- 3.4 Materials and methods -- 3.4.1 Materials -- 3.4.2 Mutagenesis, protein expression and purification -- 3. 4.3 Far-UV Circular dichroism -- 3.4.4 Thioflavin T fluorescence -- 3.4.5 Aggregation kinetics followed by static light scattering -- 3.4.6 Congo red staining -- 3.4.7 Enzymatic activity measurements -- 3.4.8 Equilibrium unfolding -- 3.4.9 Dynamic light scattering -- 3.4.10 Cysteine labelling -- Chapter 4 Role of p-stacking in amyloidoses -- 4.1 Aromatic residues and amyloidoses -- 4.1.1 Introduction -- 4.1.2 A possible role for p-stacking in amyloid-like aggregation -- 4.1.3 mt AcP as a model for investigating p-stacking -- 4.2 Results -- 4.2.1 Strategy employed -- 4.2.2 Phenylalanine 22 -- 4.2.3 Tyrosine 25 -- 4.2.4 Tyrosine 91 -- 4.2.5 Tyrosine 98 -- 4.2.6 Statistical analysis -- 4.3 Discussion -- 4.3.1 Aromatic residues promote amyloid aggregation of mt AcP due to their hydrophobicity and b-sheet propensity -- Contents -- 4.3.2 Aromatic residues are frequent in the cross-b core of fibrils but are not necessarily required -- 4.4 Conclusions -- 4.5 Materials and methods -- 4.5.1 Mutagenesis, protein expression and purification -- 4.5.2 Aggregation kinetics with Thioflavin T fluorescence -- 4.5.3 Data calculation -- Chapter 5 Final remarks -- 5.1 Conformational states distinct from the fully folded structure can present enzymatic activity -- 5.2 A comparison between the transition state ensembles of mt AcP and Sso AcP -- 5.3 Different regions of the sequence are involved in protein folding and amyloid-like aggregation -- 5.4 Conclusions -- Acknowledgements -- Appendix A Equations and formulas -- Appendix B Models for Sso AcP aggregation -- Bibliography.