Joseph Cotruvo
Summer 2005 CEBIC Fellowship
Prof. Edward Stiefel
During my CEBIC fellowship, I investigated the structure of the iron oxide core of the Dps
protein from the marine cyanobacterium Trichodesmium erythraeum in the laboratory of Prof.
Edward Stiefel, Princeton University. Gaining an understanding of the iron oxide cores of Dps
proteins may shed light on the mechanism and purpose of iron incorporation by the protein.
Iron is an essential element for nearly all organisms, and the proteins in the ferritin family
(including Dps) are utilized by these organisms for iron storage and protection from damage
from oxidizing agents. Ferritins store iron in the form of an iron oxide in the hollow centers of
their spherical cage structures. So-called “classic” ferritins can hold up to ~4,500 iron atoms,
bacterioferritins can hold up to ~2,000 iron atoms, and Dps proteins, found in bacteria and
archaea, can hold up to ~500 iron atoms. Cores of ferritins also include phosphate to varying
extents; in general, the lower the iron to phosphate ratio, the more disorganized the core.
Dps proteins accumulate in bacteria and archaea under oxidative stress conditions, and they bind
to DNA to protect it from oxidative damage, a function from which they derive their name
(DNA-binding proteins from starved cells). The structures of the iron oxide cores of classic
ferritins and, to a lesser extent, bacterioferritins have been studied extensively, and are thought to
be similar to the iron oxide mineral ferrihydrite (5Fe2O3•9H2O), a relatively amorphous, unstable
iron oxide. However, very little is known about the cores of Dps proteins.
The first stage of my summer research involved improving the protocol that our laboratory uses
to isolate and purify the Trichodesmium Dps protein. Briefly, that protocol overexpresses the
Trichodesmium Dps in E. coli into which the gene encoding the protein has been cloned.
Exploiting the extraordinary thermal stability of ferritin family proteins, and especially Dps
proteins, the protocol also includes a 10 minute heat step at 70 ˚C to purify the protein by
denaturing any other proteins present. Abhinav Jha, a sophomore in the Princeton Chemistry
Department with whom I was working this summer, and I found that this procedure did not
produce sufficiently pure protein. We initially attempted to precipitate the Dps with Zn2+ and
resuspend it (analogously to a recrystallization procedure for ferritin). However, we abandoned
experiments with this technique when we found that an additional 30 minute heating at 90 ˚C is
an easy and effective purification technique for Dps.
In the denaturing gel shown below (Fig. 1), the left six lanes show the results of the 90 ˚C
heating experiments (the six lanes at right are from precipitation experiments that I will not
discuss further). In the first lane is the Dps purified by the standard protocol discussed above,
without a 90 ˚C heat step. Lanes 2-5 contain Dps that was further purified by heating at 90 ˚C
for 30, 60, 90, and 120 minutes, respectively. The sixth lane is a standard. Lanes 1-5 show the
Dps subunit (~20 kDa) at the bottom of the gel and the removal by the heating of the majority of
the contaminants present in the sample in Lane 1. One of the higher molecular weight bands
(~40 kDa) is likely a dimer of Dps subunits. We compared this 90 ˚C heat-purified Dps with
Dps purified by fast performance liquid chromatography (FPLC) and found no significant
difference in purity.
Fig. 1. SDS-PAGE gel demonstrating the utility of the 90 ˚C heat step for Trichodesmium Dps
purification. The gel is 10% Bis-Tris and was run using MOPS running buffer.
In order to ensure that the 90 ˚C heating was not denaturing the Dps, we ran a native gel (Fig. 2).
Comparison with the ferritin, apoferritin, and BSA standards and the FPLC-purified Dps
demonstrated that the further heating did not denature the Dps and thus was a useful technique
for Trichodesmium Dps purification, with the advantage of greater ease over the Dps purification
procedure involving FPLC.
Fig. 2. 18% Tris-glycine native gel. Lane 1: ferritin. Lane 2: apoferritin. Lanes 3, 4: FPLC purified
Dps. Lanes 5, 6: Heat purified Dps sample. Lanes 7, 8: BSA.
After we obtained the pure Dps, we were able to load the protein with iron by incubation of the
apoprotein with ferrous sulfate and hydrogen peroxide. We used the ferrozine and
phenanthroline assays to quantify iron, exploring the relative pros and cons of each assay in
preparation for future studies in which the average number of iron atoms per Dps protein will
need to be quantified.
I will be continuing the research initiated in my CEBIC fellowship for my senior thesis this year.
The questions I hope to answer in my studies of the Trichodesmium Dps iron core include:
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What is the iron oxide phase?
What is the degree of structural order in the core?
Does the presence of phosphate affect this order/disorder?
Where is the phosphate located – on the core surface, in the core interior, or both?
How much iron and phosphate is present in each core?
Are the in vitro results representative of the in vivo core structure and iron and phosphate
content?
In order to answer these questions, I plan to use Raman spectroscopy, powder x-ray
diffractometry, x-ray absorption spectroscopy (including EXAFS), Mössbauer spectroscopy, and
magnetic susceptibility determination by the Evans NMR method, among other physical
methods. This summer, I performed preliminary powder x-ray diffraction studies of
recrystallized ferritin, but the core was too amorphous to give a good pattern using the x-ray
source available (Cu Kα); in the future, the use of other x-ray sources will be attempted.
Preliminary Raman studies using a ferrihydrite suspension were also carried out, in preparation
for future studies of Dps. However, we were not able to find a suitable wavelength that could be
used to study the core, although we will continue searching for one. Finally, I hope to expand
the scope of this project to the study of the core structures of other Dps proteins, especially the E.
coli Dps.