Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen鈥揇euterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation

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• 作者：Signe T. Seger ; Jens Breinholt ; Johan H. Faber ; Mette D. Andersen ; Charlotte Wiberg ; Christine B. Schj酶dt ; Kasper D. Rand
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：June 16, 2015
• 年：2015
• 卷：87
• 期：12
• 页码：5973-5980
• 全文大小：467K
• ISSN：1520-6882

文摘

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鈥揹euterium 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 伪-helix quite distant from the mutation sites, but also rendering a part of the 伪-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.