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

Kurniawan, ND; O'Leary, JM; Thämlitz, Ann-Marie LU ; Sofair, R; Werner, JM; Stenflo, Johan LU and Downing, AK (2004) In Biochemistry 43(29). p.9352-9360
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 I and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic... (More)
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 I 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 [H-1]-N-15 heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF34 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
43
issue
29
pages
9352 - 9360
publisher
The American Chemical Society
external identifiers
  • wos:000222964900007
  • pmid:15260478
  • scopus:3242716711
ISSN
0006-2960
DOI
10.1021/bi0492105
language
English
LU publication?
yes
id
2ec1a89d-efa8-40b9-bf14-6c66c864c852 (old id 272217)
date added to LUP
2007-10-23 20:02:14
date last changed
2017-01-01 05:18:10
@article{2ec1a89d-efa8-40b9-bf14-6c66c864c852,
  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 I 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 [H-1]-N-15 heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF34 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.},
  author       = {Kurniawan, ND and O'Leary, JM and Thämlitz, Ann-Marie and Sofair, R and Werner, JM and Stenflo, Johan and Downing, AK},
  issn         = {0006-2960},
  language     = {eng},
  number       = {29},
  pages        = {9352--9360},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {N-terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules},
  url          = {http://dx.doi.org/10.1021/bi0492105},
  volume       = {43},
  year         = {2004},
}