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DER VERLAG IST IN DER ZEIT VOM 12.06.2019 BIS 23.06.2019 AUSCHLIESSLICH PER EMAIL ERREICHBAR.
aktualisiert am 13. Juni 2019
978-3-8439-0283-0, Reihe Physikalische Chemie
Single-molecule fluorescence studies of Protein Folding and Molecular Chaperones
224 Seiten, Dissertation Ludwig-Maximilians-Universität München (2011), Hardcover, A5
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. 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 of Myoglobin is used as a model system of a protein folding intermediate. It is shown that it 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 acidic structures generated by molecular dynamics simulations reveals potential candidates involved in the aggregation processes of myoglobin in the acidic compact state.
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. The mitochondrial heat shock protein 70 (Hsp70), Ssc1, has a defined conformation in the ATP state with 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, 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. These differences may reflect tuning of Ssc1 to meet specific functions.
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. 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.