Single-molecule spectroscopy: investigations of protein folding to multi-laboratory consistencies on proteins

Agam, Ganesh Namdeo (2021): Single-molecule spectroscopy: investigations of protein folding to multi-laboratory consistencies on proteins. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The investigation of complex biological processes has been challenging and require a variety of sophisticated tools to interpret the underlying processes. The study of the folding process in proteins is one of the focuses of this thesis work. To this end, both spontaneous and chaperone- assisted folding mechanisms were investigated. Single-molecule fluorescence spectroscopy has been extensively applied to the study of biomolecular bindings, conformational changes, and their dynamics due to its high sensitivity, time resolution, and its ability to differentiate between homogenous and heterogenous populations. Specifically, single-molecule Förster Resonance Energy Transfer (smFRET) studies on protein folding have elucidated the basic mechanisms of spontaneous protein folding, and properties of the chaperone-substrate interactions. The possibility to measure at low concentrations making it possible to avoid the aggregation, which is difficult to avoid in ensemble experiments. To investigate the spontaneous folding mechanisms in large multi-domain proteins, two-color smFRET studies were carried out on a slowly folding version of the two-domain Maltose- binding protein (MBP). Three-color smFRET, an extension of typical two-color smFRET to three-colors, was applied on specifically labeled MBP to visualize the co-ordination between the domains as they fold. Chaperone-substrate interactions are crucial to process the substrates and thus enable them to carry out their physiological function. Cavity confinement effect of GroEL/ES, a bacterial Hsp60 on MBP folding landscape was demonstrated. Another substrate protein, p53-DNA-binding domain was probed concerning the combined action of Hsp70 and Hsp90 chaperone on its folding. To conclude the thesis work, a smFRET comparison study on proteins involving 16 laboratories was undertaken to assess the accuracy and precision of smFRET measurements as well as to determine a detection limit for dynamic motions in proteins.

• 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: 21. Juni 2021
• 1. Bericht­erstatter:in: Lamb, Don C.