An electron microscopic study of the archaeal feast/famine regulatory protein.

Abstract

Histidine residues added to the N-terminus of a polypeptide (i.e. a His-tag) was used, for the first time to our knowledge, for electron labeling of the protein upon its electron spectroscopic imaging. Originally such a His-tag was developed by another group to purify modified proteins by taking advantage of their affinity to nickel. The feast/famine regulatory protein pot0434017 (FL11) was modified by adding six His residues to its N-terminus, so that each His pair would chelate a nickel ion. An electron microscope was operated at 200 KeV, and the electrons that lost the energy by ~875 eV upon interaction with the metal were selectively focused. The majority, 60–70%, of the spots detected in the electron micrographs were paired by distances shorter than 80 Å, and over 70% of them were paired by distances shorter than 40 Å. It is concluded that the protein molecules formed dimers, and the termini of most of the protein molecules were labeled with nickel by this method.

An electron microscopic study of the archaeal feast/famine regulatory protein 4. Estimation of the particle size.

“…6a, b). If the object is not totally circular but, in fact, closer to a square, as is the case for the A form of DM1, 4) the minimum is found at 1.41/d, where d is the diagonal length (Fig. 6c).…”

“…The protein DM1 was expressed, purified, and prepared in two forms as has been described. 4), 14) In one of these forms, here referred to as the assembled (A) form, the protein is stabilized into octamers, similar to those of FL11, by interaction with a ligand present in E. coli cells. In the other form, here referred to as the disassembled (D) form, the ligand is removed and the octamers are disassembled into smaller assemblies.…”

Binding of the feast/famine regulatory protein (FFRP) FL11 (pot0434017) to DNA in the "promoter to coding" region of gene fl11

“…8d, e) functional differences between the promoter-FFRP combinations. In order to confirm our assignment of the sequence ideal for interaction with FL11, we calculated the number of bases matching with the sequence, [TGAAAAWTTTTCA]N 8 [TGAAAAWTTTTCA] at the two 13 bp ends, by scanning the genomic DNA sequence (Fig. 9).…”

Effects of amino acids on assembling of an archaeal feast/famine regulatory protein, DM1 (pot1216151)

“…In fact, in the crystal and also in the original solution, the protein is in complex with a yet unidentified ligand, which is present inside E. coli cells. 9) When the ligand is removed by hydrophobic chromatography, 8) peak 1 decreases and other peaks 2-4 increase, showing formation of smaller assemblies (Fig. 1a, b, fraction D).…”

“…Protein DM1 was expressed, purified, and prepared into the two fractions as has been described. 8) Analytical gel filtration was car-ried out using a matrix (Protein Pak 125, Waters) with the flow rate 1 ml/min. Either fraction, 10 µl, containing the protein (~1 mg/ml) and NaCl (200 mM) in 50 mM Tris-HCl buffer (pH = 7.0), was injected into the matrix, except for an experiment where a larger volume, 20 µl, of the D fraction was used (Fig.…”