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.
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Abbreviations
- Fadd-DD:
-
C-terminal domain of the Fas-associated death domain
- CARD:
-
Caspase recruitment domain
- CD:
-
Circular dichroism
- HSQC:
-
Heteronuclear single quantum coherence
- DTT:
-
Dithiothreitol
- HX:
-
Hydrogen–deuterium exchange
- GndHCl:
-
Guanidine hydrochloride
- MES:
-
2-(N-morpholino) ethanesulfonic acid
- NMR:
-
Nuclear magnetic resonance
- UV:
-
Ultraviolet
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Acknowledgments
We express our sincere thanks to Professor Paul Driscoll (University College London) for donation of complementary DNA (cDNA) encoding Fadd-DD, and to John Sonewald (Bio-Logic USA) for technical assistance with the quenched-flow studies. We are grateful to Susan Hatcher (COSMIC Facility, Old Dominion University) for the NMR time. The research was supported by a Jeffress Research Grant (J-889) from the Thomas and Kate Miller Jeffress Memorial Trust and funding from the Old Dominion University Office of Research (to L.H.G.) and an Old Dominion University Graduate Student Fellowship (to H.L.).
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L. H. Greene and H. Li contributed equally.
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Greene, L.H., Li, H., Zhong, J. et al. Folding of an all-helical Greek-key protein monitored by quenched-flow hydrogen–deuterium exchange and NMR spectroscopy. Eur Biophys J 41, 41–51 (2012). https://doi.org/10.1007/s00249-011-0756-6
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DOI: https://doi.org/10.1007/s00249-011-0756-6