Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation
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Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation. / Seger, Signe T; Breinholt, Jens; Faber, Johan H; Andersen, Mette D; Wiberg, Charlotte; Schjødt, Christine B; Rand, Kasper D.
In: Analytical Chemistry, Vol. 87, No. 12, 16.06.2015, p. 5973-80.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation
AU - Seger, Signe T
AU - Breinholt, Jens
AU - Faber, Johan H
AU - Andersen, Mette D
AU - Wiberg, Charlotte
AU - Schjødt, Christine B
AU - Rand, Kasper D
PY - 2015/6/16
Y1 - 2015/6/16
N2 - Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.
AB - Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.
U2 - 10.1021/ac504782v
DO - 10.1021/ac504782v
M3 - Journal article
C2 - 25978680
VL - 87
SP - 5973
EP - 5980
JO - Industrial And Engineering Chemistry Analytical Edition
JF - Industrial And Engineering Chemistry Analytical Edition
SN - 0003-2700
IS - 12
ER -
ID: 142475309