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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 has 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]. A novel quenched-flow H/D exchange method is now being used to detect the formation of individual hydrogen bonds in early folding intermediates of cytochrome c and staphylococcal nuclease, which accumulate in less than 100 μs. The results indicate that the initial compaction of the protein involves specific formation of secondary and tertiary structure rather than a non-specific hydrophobic collapse.

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. In collaboration with Peter N. Walsh at Temple University we recently showed that zymogen activation of factor XI (triggered by cleavage of a scissile bond) is accompanied by a major change in overall shape of this large multi-domain protein [Samuel et al, 2007]. The findings serve as a paradigm for understanding the role of protein structural rearrangements in regulating protein interactions and enzymatic activity. Other studies focus on the signaling adaptor protein Na+/H+ exchanger regulatory factor 1 (NHERF1), which plays a central role in cellular pathways relating receptors and ion channels with the actin cytoskeleton. NHERF1 contains two globular domains belonging to the PDZ domain family and a C-terminal ezrin binding (EB) motif, as well as two long segments predicted to be intrinsically disordered. We have recently shown, using NMR, circular dichroism and fluorescence methods, that NHERF1 can assume two alternative closed conformations in which the second PDZ domain engages the C-terminal region via eith non-specific interactions with the disordered linker or specific contacts with the EB motif [Cheng et al., 2009]. The initially unstructured EB region becomes helical when bound to the second PDZ domain. Together with equilibrium unfolding and ligand binding data, the findings indicate that the activity of NHERF as a signaling adaptor is regulated by a subtle balance between competing intra- and intermolecular domain-domain interactions.

1. Xu M, Beresneva O, Rosario R, Roder H. Microsecond Folding Dynamics of Apomyoglobin at Acidic pH. J Phys Chem B. 2012; 116(23):7014-25. PubMed
2. Korendovych I V, Kulp DW, Wu Y, Cheng H, Roder H, DeGrado WF. Design of a switchable eliminase. Proc Natl Acad Sci U S A. 2011;108:6823-7. PubMed
3. Chen KC, Xu M, Wedemeyer WJ, Roder H. Microsecond unfolding kinetics of sheep prion protein reveals an intermediate that correlates with susceptibility to classical scrapie. Biophys J. 2011;101:1221-30. PubMed
4. Lau WL, DeGrado WF, Roder H. The effects of pKa tuning on the thermodynamics and kinetics of folding: Design of a solvent-shielded carboxylate pair at the “a”position of a coiled coil. Biophys J. 2010; 99:2299-2308. PubMed
5. Alves C, Cheng H, Roder H, Taylor J. Intrinsic disorder and oligomerization of the hepatitis delta virus antigen. Virology. 2010; 407:333-340. PubMed
6. Cheng H, Li J, Fazlieva R, Dai Z, Bu Z, Roder H. Autoinhibitory interactions between the PDZ2 and C-terminal domains in the scaffolding protein NHERF1. Structure. 2009;17(5):660-669. PubMed
7. Latypov RF, Maki K, Cheng H, Luck SD, Roder H. Folding mechanism of reduced Cytochrome c: equilibrium and kinetic properties in the presence of carbon monoxide. J Mol Biol. 2008;383(2):437-53. PubMed
8. Maki K, Cheng H, Dolgikh DA, Roder H. Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods. J Mol Biol. 2007;368(1):244-55. PubMed
9. Samuel D, Cheng H, Riley PW, Canutescu AA, Nagaswami C, Weisel JW, Bu Z, Walsh PN, Roder H. Solution structure of the A4 domain of factor XI sheds light on the mechanism of zymogen activation. Proc Natl Acad Sci USA. 2007;104(40):15693-8. PubMed
10. Apetri AC, Maki K, Roder H, Surewicz WK. Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments. J Am Chem Soc. 2006;128(35):11673-8. PubMed
11. Latypov RF, Cheng H, Roder NA, Zhang J, Roder H. Structural characterization of an equilibrium unfolding intermediate in cytochrome c. J Mol Biol. 2006;357(3):1009-25. PubMed
12. Roder H, Maki K, Cheng H. Early events in protein folding explored by rapid mixing methods. Chem Rev. 2006;106(5):1836-61. PubMed
13. Kuwata K, Matumoto T, Cheng H, Nagayama K, James TL, Roder H. NMR-detected hydrogen exchange and molecular dynamics simulations provide structural insight into fibril formation of prion protein fragment 106-126. Proc. Natl Acad Sci USA. 2003;100(25):14790-5. PubMed