TY  - JOUR
AU  - Margetić, Aleksandra
AU  - Nannemann, David
AU  - Meiler, Jens
AU  - Huster, Daniel
AU  - Theisgen, Stephan
PY  - 2014
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/1441
AB  - GCAPs are neuronal Ca2+-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated. membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but H-2-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca2+-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca2+ and membrane mimetics.
PB  - Elsevier
T2  - Biochimica et Biophysica Acta-Biomembranes
T1  - Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles
VL  - 1838
IS  - 11
SP  - 2767
EP  - 2777
DO  - 10.1016/j.bbamem.2014.07.012
ER  - 

@article{
author = "Margetić, Aleksandra and Nannemann, David and Meiler, Jens and Huster, Daniel and Theisgen, Stephan",
year = "2014",
abstract = "GCAPs are neuronal Ca2+-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated. membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but H-2-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca2+-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca2+ and membrane mimetics.",
publisher = "Elsevier",
journal = "Biochimica et Biophysica Acta-Biomembranes",
title = "Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles",
volume = "1838",
number = "11",
pages = "2767-2777",
doi = "10.1016/j.bbamem.2014.07.012"
}

Margetić, A., Nannemann, D., Meiler, J., Huster, D.,& Theisgen, S.. (2014). Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles. in Biochimica et Biophysica Acta-Biomembranes
Elsevier., 1838(11), 2767-2777.
https://doi.org/10.1016/j.bbamem.2014.07.012

Margetić A, Nannemann D, Meiler J, Huster D, Theisgen S. Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles. in Biochimica et Biophysica Acta-Biomembranes. 2014;1838(11):2767-2777.
doi:10.1016/j.bbamem.2014.07.012 .

Margetić, Aleksandra, Nannemann, David, Meiler, Jens, Huster, Daniel, Theisgen, Stephan, "Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles" in Biochimica et Biophysica Acta-Biomembranes, 1838, no. 11 (2014):2767-2777,
https://doi.org/10.1016/j.bbamem.2014.07.012 . .

TY  - JOUR
AU  - Anđelković, Uroš
AU  - Theisgen, Stephan
AU  - Scheidt, Holger A.
AU  - Petkovic, Marijana
AU  - Huster, Daniel
AU  - Vujčić, Zoran
PY  - 2012
UR  - https://cer.ihtm.bg.ac.rs/handle/123456789/1014
AB  - Understanding the effect of surface charge on the stability of proteins is one prerequisite for "tailoring" proteins with increased thermal stability. Here, we investigated the origin of the altered thermal stability observed between the four recently isolated isoforms (EINV1-EINV4) of external invertase. External invertase from yeast Saccharomyces cerevisiae, a homodimeric glycoprotein, represents a widely used model for studying the influence of the glyco component on protein stability. The stability of the four isoforms of invertase decreases from EINV1 to EINV4, which is accompanied by an increase in negative surface charge density. Mass spectrometry analysis revealed that the isoforms share identical protein parts indicating that the differences in stability are the result of post-translational modifications. P-31 NMR analysis revealed that the isoforms contain negatively charged phosphate groups in diester and monoester forms attached to the glycan part. The total amount of phosphate bound to the polymannan component varies between the different isoforms. These results, together with the analysis of the amount of polymannan components, show that negative surface charge density does not entirely depend on the amount of phosphate but rather on its distribution. This suggests that charged groups bound to the glyco-component of a protein can influence the stability of glycoproteins.
PB  - Elsevier France-Editions Scientifiques Medicales Elsevier, Paris
T2  - Biochimie
T1  - The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density
VL  - 94
IS  - 2
SP  - 510
EP  - 515
DO  - 10.1016/j.biochi.2011.08.020
ER  - 

@article{
author = "Anđelković, Uroš and Theisgen, Stephan and Scheidt, Holger A. and Petkovic, Marijana and Huster, Daniel and Vujčić, Zoran",
year = "2012",
abstract = "Understanding the effect of surface charge on the stability of proteins is one prerequisite for "tailoring" proteins with increased thermal stability. Here, we investigated the origin of the altered thermal stability observed between the four recently isolated isoforms (EINV1-EINV4) of external invertase. External invertase from yeast Saccharomyces cerevisiae, a homodimeric glycoprotein, represents a widely used model for studying the influence of the glyco component on protein stability. The stability of the four isoforms of invertase decreases from EINV1 to EINV4, which is accompanied by an increase in negative surface charge density. Mass spectrometry analysis revealed that the isoforms share identical protein parts indicating that the differences in stability are the result of post-translational modifications. P-31 NMR analysis revealed that the isoforms contain negatively charged phosphate groups in diester and monoester forms attached to the glycan part. The total amount of phosphate bound to the polymannan component varies between the different isoforms. These results, together with the analysis of the amount of polymannan components, show that negative surface charge density does not entirely depend on the amount of phosphate but rather on its distribution. This suggests that charged groups bound to the glyco-component of a protein can influence the stability of glycoproteins.",
publisher = "Elsevier France-Editions Scientifiques Medicales Elsevier, Paris",
journal = "Biochimie",
title = "The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density",
volume = "94",
number = "2",
pages = "510-515",
doi = "10.1016/j.biochi.2011.08.020"
}

Anđelković, U., Theisgen, S., Scheidt, H. A., Petkovic, M., Huster, D.,& Vujčić, Z.. (2012). The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density. in Biochimie
Elsevier France-Editions Scientifiques Medicales Elsevier, Paris., 94(2), 510-515.
https://doi.org/10.1016/j.biochi.2011.08.020

Anđelković U, Theisgen S, Scheidt HA, Petkovic M, Huster D, Vujčić Z. The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density. in Biochimie. 2012;94(2):510-515.
doi:10.1016/j.biochi.2011.08.020 .

Anđelković, Uroš, Theisgen, Stephan, Scheidt, Holger A., Petkovic, Marijana, Huster, Daniel, Vujčić, Zoran, "The thermal stability of the external invertase isoforms from Saccharomyces cerevisiae correlates with the surface charge density" in Biochimie, 94, no. 2 (2012):510-515,
https://doi.org/10.1016/j.biochi.2011.08.020 . .