Heinrich Roder Lab
 

A central theme of our research concerns the early stages of protein folding, which are critical for understanding how the native structure of a protein, and ultimately its function, are encoded in the amino acid sequence. The insight gained not only provides a basis for protein structure prediction and de novo design, but also contributes to our mechanistic understanding and treatment of a wide range of diseases that involve aggregation of denatured or misfolded proteins. The stability and folding dynamics of proteins also have major implications with respect to understanding of the physiological consequences of mutations, in vivo folding and other cellular processes, such as trafficking and degradation. We study the dynamics of protein folding on a microsecond time scale by coupling advanced mixing techniques with various detection methods, including fluorescence and H/D exchange labeling experiments with NMR detection. In combination with protein engineering, these approaches have provided detailed insight into the folding mechanisms of a diverse set of proteins [reviewed in Roder et al., 2006].  These approaches are currently being used to elucidate the sequence determinants for folding initiation and propagation of apomyoglobin, a prototypic alpha-helical protein, as well as other model proteins. Quenched-flow H/D exchange measurements, using a novel microfludic mixing device, allow us to detect the formation of individual hydrogen bonds in early folding intermediates populated within 100 μs of refolding. Recent results indicate that the initial compaction of cytochrome c involves specific formation of secondary and tertiary structure rather than a non-specific hydrophobic collapse [Fazelinia et al., 2014].

Our group also investigates the structure, dynamics and molecular interactions of various proteins of biomedical interest in solution by using NMR spectroscopy and other biophysical methods. A recent project is aimed at understanding the structural and dynamic properties of Na⁺/H⁺ exchanger regulatory factor (NHERF), a signaling protein comprising two PDZ domains and a C-terminal ezrin binding motif, as well as two long intrinsically disordered regions. Detailed NMR and thermodynamic studies have shown that the activity of NHERF as a signaling adaptor at the membrane-cytoskeleton interface is regulated by a complex equilibrium between various open and closed conformations. Two distinct closed states of NHERF are stabilized either by specific interactions between the second PDZ domain and the C-terminus, or by non-specific interactions involving a 90-residue flexible linker. In contrast to other PDZ domains, which generally recognized C-terminal residues in an extended conformation, the initially unstructured ezrin binding region of NHERF becomes helical when bound to the PDZ domain. Our current efforts are directed at understanding the impact of structural dynamics and order-disorder transitions on the functional interactions of NHERF, as well as the role of NHERF in modulating the activity and endocytosis of the epidermal growth factor receptor.