4.6 Article

Exploring the effect of tethered domains on the folding of Grb2 protein

Journal

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 731, Issue -, Pages -

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2022.109444

Keywords

Kinetics; Multidomain folding; Misfolding; SH2; SH3

Funding

  1. Italian Ministero dell'Is-truzione dell'Universita e della Ricerca (Progetto di Interesse Invecchiamento
  2. Sapienza University of Rome [RP11715C34AEA C9B, RM1181641C2C24B9, RM11916B414C897E, RG12017297F A7223, AR22117A3CED340A]
  3. Institut Pasteur Paris [IG 24551]
  4. ACIP grant
  5. Associazione Italiana per la Ricerca sul Cancro [860517]
  6. Regione Lazio (Progetti Gruppi di Ricerca LazioInnova) [RP11715C34AEA C9B]
  7. European Union [RM1181641C2C24B9]
  8. Istituto Pasteur Italia, Teresa Ariaudo Research Project
  9. Research Program 2022-2023 Under 45 Call 2020
  10. [ACIP 485-21]
  11. [A0375-2020-36,559]

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In this study, the folding properties of the multidomain protein Grb2 were analyzed, revealing the interaction between the SH2 and C-SH3 domains during folding. These findings contribute to a better understanding of multidomain protein folding.
Two thirds of eukaryotic proteins have evolved as multidomain constructs, and in vivo, domains fold within a polypeptide chain, with inter-domain interactions possibly crucial for correct folding. However, to date, most of the experimental folding studies are based on domains in isolation. In an effort to better understand multidomain folding, in this work we analyzed, through equilibrium and kinetic folding experiments, the folding properties of the Growth factor receptor-bound protein 2 (Grb2), composed by one SRC homology 2 domain flanked by two SRC homology 3 domains. In particular we compared the kinetic features of the multidomain construct with the domains expressed in isolation. By performing single and double mixing folding experiments, we demonstrated that the folding of the SH2 domain is kinetically trapped in a misfolded intermediate when tethered to the C-SH3. Importantly, within the multidomain construct, misfolding occurred independently if refolding is started with C-SH3 in its unfolded or native state. Interestingly, our data reported a peculiar scenario, in which SH2 and C-SH3 domain reciprocally and transiently interact during folding. Altogether, the analysis of kinetic folding data provided a quantitative description of the multidomain folding of Grb2 protein, discussed under the light of previous works on multidomain folding.

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