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N-terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules

N-terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules
N-terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules
Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [1H]-15N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.
0006-2960
9352-9360
Kurniawan, Nyoman D.
ad5d4925-a260-42df-b19d-2e61c62d7a1c
Leary, Joanne M.
7a427ae7-1fb6-47fc-9150-eee1ff53c106
Thamlitz, Ann-Marie
3908d549-699e-47ee-bf5a-89a506129554
Sofair, Raphael
ad2ff402-0e61-4198-a251-2b51f18515ed
Werner, Jörn M.
1b02513a-8310-4f4f-adac-dc2a466bd115
Stenflo, Johan
34b61f72-5c8d-43bf-b006-73376871a500
Downing, A. Kristina
1e9f9050-15ea-43e7-ab29-ed301d8bc0c3
Kurniawan, Nyoman D.
ad5d4925-a260-42df-b19d-2e61c62d7a1c
Leary, Joanne M.
7a427ae7-1fb6-47fc-9150-eee1ff53c106
Thamlitz, Ann-Marie
3908d549-699e-47ee-bf5a-89a506129554
Sofair, Raphael
ad2ff402-0e61-4198-a251-2b51f18515ed
Werner, Jörn M.
1b02513a-8310-4f4f-adac-dc2a466bd115
Stenflo, Johan
34b61f72-5c8d-43bf-b006-73376871a500
Downing, A. Kristina
1e9f9050-15ea-43e7-ab29-ed301d8bc0c3

Kurniawan, Nyoman D., Leary, Joanne M., Thamlitz, Ann-Marie, Sofair, Raphael, Werner, Jörn M., Stenflo, Johan and Downing, A. Kristina (2004) N-terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules. Biochemistry, 43 (29), 9352-9360. (doi:10.1021/bi0492105).

Record type: Article

Abstract

Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [1H]-15N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.

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More information

Submitted date: 20 April 2004
Published date: 1 July 2004

Identifiers

Local EPrints ID: 56241
URI: http://eprints.soton.ac.uk/id/eprint/56241
ISSN: 0006-2960
PURE UUID: 775454a6-c8d9-49ff-a986-978e7012a60b
ORCID for Jörn M. Werner: ORCID iD orcid.org/0000-0002-4712-1833

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Date deposited: 06 Aug 2008
Last modified: 16 Mar 2024 03:36

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Contributors

Author: Nyoman D. Kurniawan
Author: Joanne M. Leary
Author: Ann-Marie Thamlitz
Author: Raphael Sofair
Author: Jörn M. Werner ORCID iD
Author: Johan Stenflo
Author: A. Kristina Downing

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