Function and solution structure of the Arabidopsis thaliana RALF8 peptide.
Ronnie O FrederickMiyoshi HarutaMarco TonelliWoonghee LeeGabriel CornilescuClaudia C CornilescuMichael R SussmanJohn L MarkleyPublished in: Protein science : a publication of the Protein Society (2020)
We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine-rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well-resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N-terminal His-tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with 15 N and 13 C for NMR analysis and obtained near complete 1 H, 13 C, and 15 N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges.
Keyphrases
- magnetic resonance
- solid state
- escherichia coli
- high resolution
- arabidopsis thaliana
- contrast enhanced
- multidrug resistant
- mass spectrometry
- signaling pathway
- klebsiella pneumoniae
- tyrosine kinase
- loop mediated isothermal amplification
- density functional theory
- binding protein
- living cells
- single molecule
- quantum dots
- pi k akt
- liquid chromatography