Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones

Sikor, Martin (2011): Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Folding of newly synthesized proteins is an essential part of protein biosynthesis and misfolding can result in protein aggregation which can also lead to several severe diseases. Protein folding is a highly heterogeneous process and rarely populated intermediate states may play an important role. Single-molecule techniques are ideally suited to resolve these heterogeneities. In this thesis, I have employed a variety of single-molecule fluorescence spectroscopy techniques to study protein folding using model systems on different levels of complexity. The acidic compact state (A state) of Myo- globin is used as a model system of a protein folding intermediate and is studied by a combination of molecular dynamics (MD) simulations and several fluorescence spectroscopic techniques. Using two-focus fluorescence correlation spectroscopy (FCS), it is shown that the A state is less compact than the native state of myoglobin, but not as expanded as the fully unfolded state. The analysis of exposed hydrophobic regions in the acidic structures generated by the MD simulations reveals poten- tial candidates involved in the aggregation processes of myoglobin in the acidic compact state. These results contribute to the understanding of disease-related fibril formation which may lead ultimately to better treatments for these diseases. A huge machinery of specialized proteins, the molecular chaperones, has evolved to assist protein folding in the cell. Using single molecule fluorescence spectroscopy, I have studied several members of this machinery. Single-pair fluorescence resonance energy transfer (spFRET) experiments probed the conformation of the mitochondrial heat shock protein 70 (Hsp70), Ssc1, in different stages along its functional cycle. Ssc1 has a very defined conformation in the ATP state with closely docked domains but shows significantly more heterogeneity in the presence of ADP. This heterogeneity is due to binding and release of ADP. The nucleotide-free state has less inter-domain contacts than the ATP or ADP-bound states. However, the addition of a substrate protein decreases the interaction between the domains even further simultaneously closing the substrate binding lid, showing that substrate binding plays an active role in the remodeling of Ssc1. This behavior is strikingly different than in DnaK, the major bacterial Hsp70. In DnaK, complete domain undocking in the presence of ADP was observed, followed by a slight re-compaction upon substrate binding. These differences may reflect tuning of Ssc1 to meet specific functions, i.e. protein import into mitochondria, in addition to protein folding. Ssc1 requires the assistance of several cofactors depending on the specific task at hand. The results of spFRET experiments suggest that the cofactors modulate the conformation of Ssc1 to enable it to perform tasks as different as protein import and protein folding. Downstream of Hsp70 in the chaperone network, the GroEL/ES complex is a highly specialized molecular machine that is essential for folding of a large subset of proteins. The criteria that distin- guish proteins requiring the assistance of GroEL are not completely understood yet. It is shown here that GroEL plays an active role in the folding of double-mutant maltose binding protein (DM-MBP). DM-MBP assumes a kinetically trapped intermediate state when folding spontaneously, and GroEL rescues DM-MBP by the introduction of entropic constraints. These findings suggest that proteins with a tendency to populate kinetically trapped intermediates require GroEL assistance for folding. The capacity of GroEL to rescue proteins from such folding traps may explain the unique role of GroEL within the cellular chaperone machinery.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 22. Dezember 2011
• 1. Bericht­erstatter:in: Lamb, Don C.