Integrated Self-Assembly of the Mms6 Magnetosome Protein to Form an Iron-Responsive Structure

Abstract

A common feature of biomineralization proteins is their self-assembly to produce a surface consistent in size with the inorganic crystals that they produce. Mms6, a small protein of 60 amino acids from Magnetospirillum magneticum strain AMB-1 that promotes the in vitro growth of superparamagnetic magnetite nanocrystals, assembles in aqueous solution to form spherical micelles that could be visualized by TEM and AFM. The results reported here are consistent with the view that the N and C-terminal domains interact with each other within one polypeptide chain and across protein units in the assembly. From studies to determine the amino acid residues important for self-assembly, we identified the unique GL repeat in the N-terminal domain with additional contributions from amino acids in other positions, throughout the molecule. Analysis by CD spectroscopy identified a structural change in the iron-binding C-terminal domain in the presence of Fe3+. A change in the intrinsic fluorescence of tryptophan in the N-terminal domain showed that this structural change is transmitted through the protein. Thus, self-assembly of Mms6 involves an interlaced structure of intra- and inter-molecular interactions that results in a coordinated structural change in the protein assembly with iron binding.

Morphological Transformations in the Magnetite Biomineralizing Protein Mms6 in Iron Solutions: A Small-Angle X-ray Scattering Study

“…Great caution has to be taken when interpreting data collected with "dry" methods such as AFM and TEM for biomacromolecules like Mms6 due to sample damage or distortion. 27 Here we report on synchrotron radiation smallangle X-ray scattering (SAXS) of Mms6 to determine its morphological characteristics in various solution conditions, as SAXS can provide an overall three-dimensional structural/ morphological information for biological macromolecular complexes in solutions. 29,30 Magnetite nanoparticles synthesized via the classical coprecipitation method by elevating the pH of a stoichiometric mixture of ferrous and ferric salts in aqueous medium at room temperature are mostly less than 20 nm and nonuniform in size.…”

“…22,25,26 Progress has been made recently in understanding the structure and properties of Mms6. 21,27 Mms6 is an amphiphilic protein with hydrophobic N-terminal and hydrophilic C-terminal, is believed to exist as a membrane protein in vivo, and selfassembles in vitro to form a multimeric micellar complex larger than 300 kDa. 19,21,27,28 The C-terminal domains bind ferric iron ions and complexes with very high affinity.…”

“…21,27 Mms6 is an amphiphilic protein with hydrophobic N-terminal and hydrophilic C-terminal, is believed to exist as a membrane protein in vivo, and selfassembles in vitro to form a multimeric micellar complex larger than 300 kDa. 19,21,27,28 The C-terminal domains bind ferric iron ions and complexes with very high affinity. 21,28 Shuffling the amino acid sequence in a protein affects its function and thus is a good test to understand the mechanism of biomineralization.…”

“…20,21 Histagged m2Mms6 was generated by shuffling the hydroxyl/carboxyl containing amino acid residues in the C-terminal domain of His-tagged Mms6, such that m2Mms6 shares the same hydropathy profile as Mms6. 21,27 For simplicity, the His-tagged Mms6 and m2Mms6 are simply referred to as Mms6 and m2Mms6 (both consisting of 99 amino acid residues, molecular weight (MW) ≈ 10 kDa, average molecular volume v mol ≈ 1.3 × 10 4 Å 3 ). 32 Mms6 and m2Mms6 were purified in inclusion bodies, refolded as previously reported, 20,21,27 and dissolved in the buffer BC100 (20 mM Tris, 100 mM KCl, pH 7.5).…”

“…21,27 For simplicity, the His-tagged Mms6 and m2Mms6 are simply referred to as Mms6 and m2Mms6 (both consisting of 99 amino acid residues, molecular weight (MW) ≈ 10 kDa, average molecular volume v mol ≈ 1.3 × 10 4 Å 3 ). 32 Mms6 and m2Mms6 were purified in inclusion bodies, refolded as previously reported, 20,21,27 and dissolved in the buffer BC100 (20 mM Tris, 100 mM KCl, pH 7.5). Proteins in BC100 were flash-frozen with liquid nitrogen and kept frozen prior to use.…”