Studies of the structure and function of Mms6, a bacterial protein that promotes the formation of magnetic nanoparticles

Thesis/Dissertation · Sat Jan 01 00:00:00 EST 2011

DOI:https://doi.org/10.2172/1029600· OSTI ID:1029600

Wang, Lijun ^[1]

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1. Iowa State Univ., Ames, IA (United States)

Here we report structural and functional studies of Mms6, a biomineralization protein that can promote the formation in vitro of magnetic nanoparticles with sizes and morphologies similar to the magnetites synthesized by magnetotactic bacteria. We found the binding pattern of Mms6 to ferric ion to be two-phase and multivalent. We quantatively determined that Mms6 binds one Fe³⁺ with a very high affinity (K_d = 10¹⁶ M). The second phase of iron binding is multivalent and cooperative with respect to iron with a K_d in the μM range and a stoichiometry of about 20 ferric ion per protein molecule. We found that Mms6 exists in large particles of two sizes, one consisting of 20-40 monomeric units and the other of 200 units. From proteolytic digestion, ultracentrifugation and liposome fusion studies, we found that Mms6 forms a large micellar quaternary structure with the N-terminal domain self-assembling into a uniformly sized micelle and the C-terminal domain on the surface. The two-phase iron-binding pattern may be relevant to iron crystal formation. We propose that the first high affinity phase may stabilize a new conformation of the C-terminal domain that allows interaction with other C-terminal domains leading to a structural change in the multimeric protein complex that enables the second low affinity iron binding phase to organize iron and initiate crystal formation. We also observed a dimeric apparent molecular mass of the Mms6 C-terminal peptide (C21Mms6). We speculate that the C-terminal domain may form higher order quaternary arrangements on the surface of the micelle or when anchored to a membrane by the N-terminal domain. The change in fluorescence quenching in the N-terminal domain with iron binding suggests a structural integrity between the C- and N-terminal domains. The slow change in trp fluorescence as a function of time after adding iron suggests a very slow conformational change in the protein that involves both N- and C-terminal domains. We interpret these results to mean that there is a coordinated global change in Mms6 structure that involves multiple Mms6 monomers. Based on our observations, we propose a mechanism by which Mms6 can promote the formation of crystalline nanoparticles. Upon binding ferric iron at very high affinity with a molar ratio of 1, the C-terminal domains undergo a conformational change, coordinated with the N-terminal domain that initiates a slow rearrangement of the multiprotein complex to create a surface on which many iron atoms can organize. This slow rearrangement allows the initiation of a crystal that is propagated on the protein surface to form a crystal seed. We also observed that Mms6 undergoes a periodical structural change with increasing molar ratios of iron to protein. The observed structural change occurs in the N-terminal domain, but reflects iron binding by the C-terminal domain. A mutant Mms6 that does not promote the formation of superparamagnetic magnetite also does not undergo the periodic structural change. The correlation between the ability to form magnetic nanoparticles and the ability to undergo the periodic structural change suggests that this structural change might participate in the mechanism of magnetite formation. Analysis of the quaternary structure of Mms6 protein and its C-terminal domain reveals that the C-terminal domain contributes to the overall stability of the quaternary structure of Mms6. The C-terminal domain of Mms6 displays ferric reductase activity that is diminished in the mutant C-terminus. Although it does not play a role in the in vitro formation of magnetite during which both ferrous and ferric forms of iron are provided, this reductase activity may participate in magnetite formation in vivo. Our results suggest that the iron binding properties and quaternary structure of Mms6 is responsible for formation of magnetic nanoparticles. The ability for Mms6 protein to form a quaternary structure and the ability of that structure to change as iron is incorporated into a crystal may be fundamental aspects of the mechanism by which a small protein such as Mms6 can promote the formation of magnetite crystals.

• Research Organization: Ames Lab., Ames, IA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• DOE Contract Number: AC02-07CH11358
• OSTI ID: 1029600
• Report Number(s): IS-T 3020; TRN: US201201%%886
• Country of Publication: United States
• Language: English

Related Subjects

36 MATERIALS SCIENCE
75 CONDENSED MATTER PHYSICS
SUPERCONDUCTIVITY AND SUPERFLUIDITY
AFFINITY
ATOMS
BACTERIA
CONFORMATIONAL CHANGES
DIGESTION
FLUORESCENCE
FUNCTIONALS
IN VITRO
IN VIVO
IRON
LIPOSOMES
MAGNETITE
MEMBRANES
MONOMERS
MUTANTS
OXIDOREDUCTASES
PEPTIDES
PROTEINS
QUENCHING
STABILITY
STOICHIOMETRY
ULTRACENTRIFUGATION