Degree Type

Dissertation

Date of Award

2015

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Molecular, Cellular, and Developmental Biology

First Advisor

Marit Nilsen-Hamilton

Abstract

Biomineralization by living organisms provides excellent examples of controlled mineral synthesis for us to learn how to produce materials with desired morphologies and properties under ambient conditions. Magnetotactic bacteria (MTBs) are an example of biomineralizing organisms that produce magnetic nanoparticles. MTBs are a diverse family of organisms that are capable of producing nano-sized magnetic particles inside the cell-body with finely controlled particle size and magnetic properties. A single protein, Mms6, from these bacteria was shown to direct the biomineralization of magnetite nanoparticles from iron solutions in vitro. Previous work showed that Mms6 forms micelles in solution, with the hydrophobic N-terminal domain incorporated in the micelle and C-terminal domain exposed on the surface of the micelle. Evidence was obtained for a structural change of Mms6 when it binds with Fe3+ as shown by CD spectroscopy and by measuring intrinsic tryptophan fluorescence. But how the protein forms micelles and undergoes structural change upon contact with iron regulates the crystallization process was still to be determined.

By using TEM and AFM microscopy, I visualized the spherical micelles formed by Mms6. The results reported in chapter 2 of this thesis 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 protein mutational studies to determine the amino acid residues important for self-assembly, I 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.

Mms6 displays two distinct types of tryptophan fluorescence spectra when tested in quartz cuvettes and plastic 96 well plates. Further investigation showed that Mms6 adsorbs onto hydrophobic plastic surfaces. In chapter 3, we report that Mms6 undergoes structural rearrangements on binding iron that can be measured by intrinsic trp fluorescence. Both phases of iron binding (high affinity stoichiometric and low affinity, high capacity) were linked to the fluorescence changes. The high affinity and stoichiometric binding measured at pH 7 demonstrated the same high affinity as was determined by direct iron binding with 55Fe filter capture assays. This fluorescence change is proposed to be an intramolecular structural change as it is not temperature-dependent. The high capacity and low affinity binding of iron is cold sensitive as is the fluorescence change that could be measured at low pH with high molar ratios of iron to protein. Trp119 was identified as the residue for which the signal was measured. Thus intrinsic fluorescence spectroscopy reveals a complex combination of structural changes in Mms6 that probably involve both intra and inter-molecular interactions.

The observation, made by our colleagues, that Mms6 binds ferric and not ferrous iron brought up the question of how Mms6 regulates the crystallization of magnetite, which contains both ferric and ferrous iron. The results, reported in Chapter 4 of this thesis, showed that Mms6 is a ferric reductase, which can catalyze the reduction of ferric iron to ferrous iron using NADH and FAD as electron donors and cofactors, respectively. Higher reductase activity was observed when Mms6 was integrated into either liposomes or bicelles. These results are consistent with a hypothesis that Mms6 is a membrane protein which promotes the formation of magnetite by a mechanism that involves reducing iron.

DOI

https://doi.org/10.31274/etd-180810-4429

Copyright Owner

Shuren Feng

Language

en

File Format

application/pdf

File Size

185 pages

Included in

Biochemistry Commons

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