Single-Molecule Spectroscopy of the Structure, Dynamics, and Folding of Proteins

Schuler1

We investigate the structure and dynamics of proteins with optical single-molecule spectroscopy. In particular, we focus on systems with pronounced conformational heterogeneity that is difficult to resolve with other techniques. Examples are intrinsically disordered proteins (IDPs), proteins interacting with molecular chaperones, protein misfolding, and the behavior of proteins inside live cells. A key goal of our work is to reach mechanistic understanding based on quantitative physical models.
 
Addressing these questions requires a broad spectrum of complementary methods, and thus a multidisciplinary team of scientists from physics, chemistry, and biology closely collaborate within the group. We use an integrative approach ranging from molecular biology and protein chemistry to a wide range of biophysical methods, single-molecule spectroscopies, and simulations. An important aspect of our research is the continuous development and adaptation of advanced single-molecule techniques and analysis methods for probing biological macromolecules over a wide range of conditions and timescales.
 
Protein folding dynamics and intrinsically disordered proteins
We study fundamental aspects that govern protein structure and dynamics in vitro and in vivo by combining the information on molecular distances and dynamics available from single molecule spectroscopy. This approach has allowed us to map intramolecular distance distributions, determine nanosecond dynamics that govern the diffusive search of a protein on its free energy surface, and investigate folding and misfolding dynamics over a wide range of timescales. Single-molecule methods are also ideally suited for probing the structure, dynamics, and functions of intrinsically disordered proteins. Key methods employed are single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET).
 
Effects of the cellular machinery on protein folding and misfolding mechanisms
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. However, our mechanistic understanding of how these cellular components affect the free energy landscape of the folding process has remained very limited, largely due to a lack of suitable methods. We investigate the role of cellular factors on protein folding mechanisms with single molecule fluorescence spectroscopy, in particular the effects of molecular chaperones. A detailed investigation of these processes will be crucial for understanding the fine balance between protein folding and misfolding in the cell, and the large number of diseases associated with protein misfolding and aggregation.
 
Single molecule spectroscopy
A wide range of single-molecule instrumentation is available in the group, including a TIRF microscope and several state-of-the-art confocal instruments with picosecond counting electronics and a large variety of laser sources. Our projects are often based on the development of novel instrumentation and data analysis tools, frequently in close combination with theory and simulations. By taking full advantage of all observables from single-photon counting, both quantitative distance information and conformational dynamics on many timescales become accessible. Examples include nanosecond reconfiguration times from rapid correlation spectroscopy and millisecond to minute kinetics from single molecule detection with microfluidic mixing devices.
 
Protein chemistry
A prerequisite for single molecule FRET experiments is the site-specific labeling of proteins with suitable fluorophores. We use modern molecular biology methods, recombinant heterologous expression, and purification with advanced chromatography techniques to generate the proteins of choice. We also develop new methods to improve specificity and versatility of fluorophore incorporation.