Computational and experimental studies focusing on the role of conserved residues for folding and stability is an active and promising area of research. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. 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. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native topology. Equilibrium unfolding and refolding studies of a model superfamily member, the Fas-associated death domain protein indicate that this process is cooperative, two-state and reversible. Stopped-flow fluorescence studies reveal that the folding is rapid and biphasic with the majority of the hydrophobic core forming in the first phase. Site-directed mutagenesis studies indicate that conserved Trp112, Trp148, Leu115 and Val121 are important to structure, native state stability and folding.

We also present the results of experiments aimed at characterizing the formation of secondary structure. Stopped-flow far-UV CD spectroscopy revealed that the folding process was monophasic and the rate is 23.4 s^-1. To gain atomic resolution a combination of quenched-flow methods, hydrogen deuterium exchange (HX) and NMR spectroscopy was implemented. 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 rates between 19 s^-1 and 22 s^-1. These results indicate that the formation of secondary structure is largely cooperative and concomitant with the hydrophobic collapse. Additional insights are gained by calculating the exchange rates of twenty-three residues from equilibrium FIX 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. These results appear to be the earliest conservation analysis and biophysical characterization conducted on the Fas-associated death domain and folding kinetics using quenched-flow combined with NMR spectroscopy on an all a-helical Greek-key protein.