title: Probing the Conformational and Functional Consequences of Disulfide Bond Engineering in Growth Hormone by Hydrogen-Deuterium Exchange Mass Spectrometry Coupled to Electron Transfer Dissociation.
abstract: 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.
authors.affiliation: †Global Research, Novo Nordisk A/S, Novo Nordisk Park 1, 2760 Måløv, Denmark.|‡Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 København Ø, Denmark.
authors.forename: Signe T
authors.initials: ST
authors.lastname: Seger
authors.affiliation: †Global Research, Novo Nordisk A/S, Novo Nordisk Park 1, 2760 Måløv, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Breinholt
authors.affiliation: †Global Research, Novo Nordisk A/S, Novo Nordisk Park 1, 2760 Måløv, Denmark.
authors.forename: Johan H
authors.initials: JH
authors.lastname: Faber
authors.affiliation: †Global Research, Novo Nordisk A/S, Novo Nordisk Park 1, 2760 Måløv, Denmark.
authors.forename: Mette D
authors.initials: MD
authors.lastname: Andersen
authors.affiliation: †Global Research, Novo Nordisk A/S, Novo Nordisk Park 1, 2760 Måløv, Denmark.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Wiberg
authors.affiliation: †Global Research, Novo Nordisk A/S, Novo Nordisk Park 1, 2760 Måløv, Denmark.
authors.forename: Christine B
authors.initials: CB
authors.lastname: Schjødt
authors.affiliation: ‡Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 København Ø, Denmark.
authors.forename: Kasper D
authors.initials: KD
authors.lastname: Rand
mesh_terms: D041961:Deuterium Exchange Measurement; D004220:Disulfides / Q000737:chemistry*; D004579:Electron Transport; D019382:Human Growth Hormone / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D004220:Disulfides; D019382:Human Growth Hormone