Studying the mitochondrial Hsp70, Ssc1, in vitro and in organello using single-molecule Förster Resonance Energy Transfer

Trauschke, Vanessa (2021): Studying the mitochondrial Hsp70, Ssc1, in vitro and in organello using single-molecule Förster Resonance Energy Transfer. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

Hsp70 proteins are molecular chaperones that stabilize unfolded proteins and are important for preventing protein aggregation. The mitochondrial Hsp70 of yeast, Ssc1, is additionally involved in translocation of proteins into the mitochondrial matrix. The functions of proteins are to a significant extent determined by their conformational dynamics and interactions with other biomolecules. Single-molecule Förster Resonance Energy Transfer (smFRET) is a widely applied and powerful method to study the conformational dynamics of proteins. We performed smFRET measurements of Ssc1 in vitro, which revealed conformational dynamics on the sub-millisecond and second timescale. Our investigations further gave new insights into the influence of nucleotides, substrate and the cochaperones Mdj1 and Mge1 on the structure and dynamic behavior of Ssc1. While in vitro experiments allow for precise control of the buffer and available interaction partners, the obtained conformational changes and kinetics might not be the same as in vivo. However, smFRET experiments in living cells remain challenging because of high background, need of low labeled sample concentrations and the challenge of getting the labeled proteins into living cells. Here, we perform in organello smFRET measurements on Ssc1 inside isolated mitochondria to get an idea about the conformation and dynamics in its natural environment while containing low background and high signal intensities. Our in organello experiments showed that most Ssc1 inside mitochondria is substrate-bound. This observation implies substantial consequences for the role of Ssc1 as a chaperone and in protein translocation into the matrix. Tracking of single Ssc1 proteins inside mitochondria further allowed us to put mobility and conformation of Ssc1 into context and assign a small fraction of substrate-free but translocase-bound Ssc1.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: Ssc1, mitochondrial Hsp70, single-molecule FRET, protein dynamics, in organello
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 25. June 2021
• 1. Referee: Lamb, Don C.
• Signature of the printed copy: 0001/UMC 28083