Final Report Summary - SINGLEMOLFOLDING (Towards Protein Folding in the Cell with Single Molecule Spectroscopy)
Many aspects of the physical principles governing protein folding in vitro have been elucidated in the past decades. At the same time, a large number of cellular components involved in protein folding in vivo have been identified. But our mechanistic understanding of how these cellular components affect the energy landscape of the folding process has remained very limited, largely due to a lack of suitable methods. An opportunity to bridge this gap is single molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET), a new technique that allows the investigation of distance distributions and dynamics of individual protein molecules even in complex and heterogeneous environments. In a multi-disciplinary project employing methods ranging from molecular biology and protein biochemistry to optics, microfluidic mixing, and development of instrumentation and software, we used single molecule spectroscopy for investigating the question how proteins find their native three-dimensional structure in a cell.
We have further developed and adapted methods and combined them to study these processes, both in terms of instrumentation and data analysis. For two chaperone systems that have been investigated, pronounced effects have been observed. Confinement in the chaperonin GroEL/ES decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. This surprising observation is probably due to the pronounced compaction of the polypeptide, which results in increased internal friction and/or interactions with the cavity walls. For the complementary chaperone system DnaK/J, we find a strong expansion of the substrate protein upon chaperone binding, which may be crucial for the downstream processes of protein folding in the cell. New method developments now also allow us to probe proteins directly in live cells, i.e. directly in their natural environment.
We have further developed and adapted methods and combined them to study these processes, both in terms of instrumentation and data analysis. For two chaperone systems that have been investigated, pronounced effects have been observed. Confinement in the chaperonin GroEL/ES decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. This surprising observation is probably due to the pronounced compaction of the polypeptide, which results in increased internal friction and/or interactions with the cavity walls. For the complementary chaperone system DnaK/J, we find a strong expansion of the substrate protein upon chaperone binding, which may be crucial for the downstream processes of protein folding in the cell. New method developments now also allow us to probe proteins directly in live cells, i.e. directly in their natural environment.