The metal binding properties of the CCCH motif of the 50S ribosomal protein L36 from Thermus thermophilus.
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).
Boysen
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RI
Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
Hearn
M T
MT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D007455:Iodine / Q000737:chemistry / Q000494:pharmacology; D013058:Mass Spectrometry; D008623:Mercaptoethanol / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012269:Ribosomal Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D016963:Thermus thermophilus / Q000737:chemistry*; D013997:Time Factors; D016335:Zinc Fingers
D008670:Metals; D010455:Peptides; D012269:Ribosomal Proteins; D008623:Mercaptoethanol; D007455:Iodine; D008628:Mercury; C029823:mercuric acetate