The metal binding properties of the CCCH motif of the 50S ribosomal protein L36 from Thermus thermophilus

Abstract

The TthL36 protein of the 50S ribosomal proteins from Thermus thermophilus has been found to contain the rare C(Xaa)2C(Xaa)12C(Xaa)4H (CCCH) sequence motif, a zinc finger binding motif, which for other zinc finger proteins is known to cleave RNA hairpins. In order to investigate the metal-binding properties of this T. thermophilus TthL36 protein, the core 26-mer polypeptide containing this CCCH motif was prepared by solid-phase peptide synthesis methods using Fmoc chemistry, purified by preparative RP-HPLC and characterized by circular dichroism, high-performance capillary zone electrophoresis and electrospray ionization mass spectrometry. Reaction of the acetamidomethyl (Acm)-protected polypeptide with iodine under acidic conditions resulted in the formation of the fully de-protected polypeptide. Of interest, the results demonstrate that the standard Acm-deprotection method with the synthetic TthL36 polypeptide using mercuric acetate in the presence of a large excess of 2-mercaptoethanol resulted in preferential formation of a very stable mercuro-polypeptide complex. The properties of the Acm-deprotected polypeptide in the presence of different metal ions were also investigated by spectroscopic methods. The results confirm that this TthL36 polypeptide containing the CCCH motif binds metal ions with different affinities, namely in the order Co(II)>Hg(II)>Zn(II).

Open‐tubular electrochromatographic characterization of synthetic peptides

“…The peptides 1-8 used in this study were prepared by solid-phase peptide synthesis based on Fmoc-chemistry [12][13][14][15]. The generic methods employed for the purification of these synthetic peptides have been described previously [14].…”

Characterization and applications of etched chemically modified capillaries for open-tubular capillary electrochromatography

“…The TRAP peptides used in this study were prepared [27] according to the general principles of solid phase peptide synthesis based on Fmoc-chemistry ( [28,29] …”

DNA Oxidative Cleavage Induced by the Novel Peptide Derivatives of 3‐(quinoxalin‐6‐yl)alanine in Combination with Cu(II) or Fe(II) Ions

“…Many recent applications of this soft ionisation technique suggest that gas phase data reflect, to some extent, the interactions occurring in solution [28]. This method is also established in the studies of metal-protein and metal-peptide interactions, providing information on the stoichiometry of complexes [29], and to a certain degree, the localization of binding sites [30, 31]. …”

“…Mechanistic investigations using distamycin revealed a minor and a major groove binding for the Cu(II)–DPQ and Cu(II)–DPPZ complexes, respectively, thus suggesting different effects exerted by both quinoxaline derivatives on DNA [29]. On this basis we have compared the influence of DPPZa-Gly (lanes 1–6, see Figure 5 in the Supplementary Material) and DPQa-Gly (lanes 7–12) on the electrophoretic mobility of the plasmid.…”