title: Folding of an all-helical Greek-key protein monitored by quenched-flow hydrogen-deuterium exchange and NMR spectroscopy.
abstract: To advance our understanding of the protein folding process, we use stopped-flow far-ultraviolet (far-UV) circular dichroism and quenched-flow hydrogen-deuterium exchange coupled with nuclear magnetic resonance (NMR) spectroscopy to monitor the formation of hydrogen-bonded secondary structure in the C-terminal domain of the Fas-associated death domain (Fadd-DD). The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all-β-sheet immunoglobulin and mixed α/β-plait superfamilies. Fadd-DD is selected as our model death domain protein system because the structure of this protein has been solved by NMR spectroscopy, and both thermodynamic and kinetic analysis indicate it to be a stable, monomeric protein with a rapidly formed hydrophobic core. Stopped-flow far-UV circular dichroism spectroscopy revealed that the folding process was monophasic and the rate is 23.4 s(-1). Twenty-two amide hydrogens in the backbone of the helices and two in the backbone of the loops were monitored, and the folding of all six helices was determined to be monophasic with rate constants between 19 and 22 s(-1). These results indicate that the formation of secondary structure is largely cooperative and concomitant with the hydrophobic collapse. This study also provides unprecedented insight into the formation of secondary structure within the highly populated Greek-key fold more generally. Additional insights are gained by calculating the exchange rates of 23 residues from equilibrium hydrogen-deuterium exchange experiments. The majority of protected amide protons are found on helices 2, 4, and 5, which make up core structural elements of the Greek-key topology.
authors: Department of Chemistry and Biochemistry, Old Dominion University, 4541 Hampton Boulevard, Norfolk, VA 23529, USA. lgreene@odu.edu
authors: Lesley H
authors: LH
authors: Greene
authors: Hai
authors: H
authors: Li
authors: Junyan
authors: J
authors: Zhong
authors: Guoxia
authors: G
authors: Zhao
authors: Khym
authors: K
authors: Wilson
mesh_terms: D041961:Deuterium Exchange Measurement; D053200:Fas-Associated Death Domain Protein / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D053200:Fas-Associated Death Domain Protein