FORCE SPECTROSCOPY OF MINERAL-MICROBE BONDS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 303

By applying the WLC model to experimentally measured force spectra, we
were able to identify two putative proteins as potential candidates involved in
the formation of a bond with Fe(III) minerals (see Fig. 14.2 ) . These proteins
had contour lengths of approximately 290 and 540 nm, which corresponded
to polypeptides with masses of approximately 80 and 150 kD, respectively.
Fortunately, at this same time, the genome of had been

determined, 50 and we had begun to use two-dimensional gel electrophoresis
to characterize the outer membrane proteins produced by

S. oneidensis

. 31,32
Partly on the basis of the whole-cell force spectra noted earlier, we decided
to focus our efforts on two outer membrane proteins from

S. oneidensis

MtrC
and OmcA. These two proteins contained heme groups, which meant they
could catalyze electron transfer reactions. Furthermore, each protein had a
molecular mass of ~80 kD, which was consistent with the putative protein
force-signatures in the AFM force spectra.
Therefore, MtrC and OmcA from

S. oneidensis:

were produced in large
enough quantities to use in the AFM. Each protein was linked to a gold
substrate. The AFM tip was coated with a thin ilm of an Fe(III) mineral, and
this mineral-coated tip was used to probe MtrC and OmcA ( Fig. 14.3 ) . The
resulting force spectra were the irst of their kind for a bacterial cytochrome
protein that formed a bond with a crystalline Fe(III) mineral. 33 Further, the
spectra for the pure protein could be compared directly with the spectra
collected on living cells of

S. oneidensis

.
Recall that the whole-cell force spectra lead us to hypothesize that proteins
of ~80 and 150 kD were responsible for the bond between

S. oneidensis

S. oneidensis and

Fe(III) minerals. However, decorates its outer surface with many

different proteins, not just the two proteins of interest. The ultimate test of
our hypothesis came when we compared the force spectra for a living cell of
S. oneidensis

S. oneidensis

with those collected on individual proteins that were puriied
from the outer membrane of .

Figure 14.3 compares the whole-cell and protein force spectra. It is
important to note that the cell and protein data were collected on two
completely different AFMs. There are signiicant similarities between the cell
and protein spectra. Speciically, there are two distinct, nonlinear, sawtooth-
shaped force-signatures at approximately 300 and 550 nm. The shorter
sawtooth observed for a living cell of

S. oneidensis

is consistent with the
forced unfolding of either MtrC or OmcA. The longer sawtooth is still a bit of a
challenge to explain. It is too long to correspond to the mechanical unfolding
of a monomer of MtrC or OmcA. However, a dimer of either protein, linked
end to end, would have a contour length of approximately the same length as
the longer sawtooth. Indeed, this same sawtooth was observed when force
spectra were collected on puriied MtrC (see Fig. 14.3 ) .

S. oneidensis