Folding of the nascent polypeptide chain of a histidine phosphocarrier protein in vitro
| Autor: | José L. Neira, Martina Palomino-Schätzlein |
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| Přispěvatelé: | Generalitat Valenciana, Neira, José L. |
| Jazyk: | angličtina |
| Rok vydání: | 2023 |
| Předmět: | |
| Popis: | 15 pags., 8 figs., 3 tabs. The phosphotransferase system (PTS), a metabolic pathway formed by five proteins, modulates the use of sugars in bacteria. The second protein in the chain is the histidine phosphocarrier, HPr, with the binding site at His15. The HPr kinase/phosphorylase (HPrK/P), involved in the bacterial use of carbon sources, phosphorylates HPr at Ser46, and it binds at its binding site. The regulator of sigma D protein (Rsd) also binds to HPr at His15. We have designed fragments of HPr, growing from its N-terminus and containing the His15. In this work, we obtained three fragments, HPr38, HPr58 and HPr70, comprising the first thirty-eight, fifty-eight and seventy residues of HPr, respectively. All fragments were mainly disordered, with evidence of a weak native-like, helical population around the binding site, as shown by fluorescence, far-ultraviolet circular dichroism, size exclusion chromatography and nuclear magnetic resonance. Although HPr38, HPr58 and HPr70 were disordered, they could bind to: (i) the N-terminal domain of first protein of the PTS, EIN; (ii) Rsd; and, (iii) HPrK/P, as shown by fluorescence and biolayer interferometry (BLI). The association constants for each protein to any of the fragments were in the low micromolar range, within the same range than those measured in the binding of HPr to each protein. Then, although acquisition of stable, native-like secondary and tertiary structures occurred at the last residues of the polypeptide, the ability to bind protein partners happened much earlier in the growing chain. Binding was related to the presence of the native-like structure around His15. This research has been funded by la Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana, Valencian Regional Government) [CIAICO 2021/0135 to JLN]. Some of the NMR equipment used in this work has been funded by Generalitat Valenciana (Spain) and co-financed with ERDF funds (OP ERDF of Generalitat Valenciana (Spain) 2014–2020). The funders did not have any role during acquisition and analyses of experiments or in the publication of this manuscript. |
| Databáze: | OpenAIRE |
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