期刊
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS
卷 1865, 期 12, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.bbagen.2021.130015
关键词
Binding; Circular dichroism; Fluorescence; Histidine-phosphocarrier-protein; NMR; Protein-protein interactions
资金
- Spanish Ministry of Economy and Competitiveness
- European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00, RTI2018-101969-J-I00]
- Generalitat Valenciana (Spain)
- ERDF funds (OP ERDF of Comunitat Valenciana (Spain)
The study focused on the design of a specific fragment of the HPr protein, HPr48, which was shown to be disordered. Despite being only 48 residues long, HPr48 was capable of binding to other proteins and aiding in antimicrobial activity of antibiotics.
Background: The phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes. Methods: We obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR). Results: Secondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules. Conclusions: Although HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity. General significance: HPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones.
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