DISSECTING STRUCTURAL ASPECTS OF PROTEIN STABILITY

The contribution of a single residue in the structural stability of a whole protein can seem small if its position in the structure is not considered. Indeed, the stability of a protein is determined by the contribution of many forces among amino acids: hydrophobicity, hydrogen-bonding network and van der Waals interactions, salt bridges. Three independent projects were carried out during my PhD studies addressing mutations that affect protein fold stability. I β2-Microglobulin (β2m), the light chain of the Major Histocompatibility Class I complex (MHC-I), can assemble into amyloid fibrils and deposit in joints and bones of patients receiving long term haemodialysis, leading to dialysis-related amyloidosis (DRA). β2m has been object of many mutational studies to investigate the molecular bases of its aggregation propensity. Among other residues, these analyses have pointed out that Trp60 plays a crucial role for β2m stability, in particular, W60G β2m, has an increased conformational stability and a reduced amyloidogenicity compared to the wild type protein. To identify the key-residues that prompt the amyloidogenicity of β2m, the native state dynamics of WT β2m and W60G β2m have been compared, combining solution NMR spectroscopy and molecular dynamics (MD) simulations. As a result, three β2m variants were designed and characterized: W60G-Y63W, W60G-N83V and V85E. All mutants have displayed the expected structure, stability and aggregation propensity, giving insights into structural determinants of aggregation propensity of WT β2m. II To date, only a single natural β2m variant at residue Asp76, D76N β2m, has been reported. This mutation destabilizes the protein making it more prone to aggregation and has been related to a systemic and hereditary amyloid disorder. To study the fundamental properties of protein dynamics and stability in solution and in crystal, D76N, WT and W60G β2m variants were chosen. These proteins display significant different stabilities and aggregation propensities, although conserving well comparable structures, with the same crystal symmetry and number of molecules in the asymmetric unit. These properties make them suitable as model system to obtain results free of spurious effects, reflecting the changes on protein dynamics related to the mutation. Thus, these three variants were used to investigate the correlation between protein stability in solution and in crystalline form by Fourier transform infrared spectroscopy (FT-IR). FT-IR spectra indicate that each β2m variant displays an increased structural order in the crystals compared to the protein in solution; temperature ramps show that increased melting temperatures for crystals compared to protein in solution. Nevertheless, the differences in protein stability between the three variants are well detectable in crystalline form and they well correlate with the trend observed in solution. III MHC-I complexes are hetero-trimers composed by the polymorphic heavy chain, the light chain β2m and an antigenic peptide. The overall structural stability of the complex together with peptide-complex affinity, is fundamental for an effective presentation of the antigen: a strong peptide/MHC-I (pMHC) interaction and a time-extended peptide presentation trigger the best immunogenic CD8+ T-cell response. Accordingly, aim of the work is the exploration of the impact on protein fold stability of complex-stabilizing point mutations on the tumour antigenic peptide NY–ESO157-165 (SLLMWITQC). This peptide is expressed by a broad range of tumour types, but not in healthy adult somatic tissue, making it an ideal cancer vaccine candidate. The in vitro stability of eight complexes has been monitored following the thermal unfolding. All complexes bearing the mutated peptides displayed an increase in stability compared to that of the complex with the NY–ESO157-165 WT. The structural contributions to the stability of the complexes have been investigated through X-ray crystallography. Thanks to the structural studies it has been possible to deepen the molecular characteristics determining peptide binding to MHC- I molecules and rationalize the optimization of the MHC- I anchor residues in tumour epitopes to enhance binding of the peptide to the complex.