SINGLEMOLECULE FORCE SPECTROSCOPY OF MICROBIAL CELL ENVELOPE PROTEINS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 321

These nanoscale measurements were shown to correlate with microscale
mycobacterial aggregation assays. Cultures of native bacteria and of a mutant
lacking HBHA were incubated with growing concentrations of HBHA and
observed with an optical microscope. The addition of HBHA to the cell
suspensions induced cellular aggregation in a dose-dependent manner.
By contrast, the mutant strain did not signiicantly aggregate following
addition of HBHA. These observations support the notion that mycobacterial
aggregation involves homophilic HBHA-HBHA interactions, measured here
for the irst time at the single-molecule level.
In summary, the data surveyed here indicate that SMFS offers new
avenues for elucidating the molecular mechanisms of bacterial adhesion, e.g.,
in the context of infection diseases, and for developing new anti-adhesion
strategies for therapy.

15.3 MECHANICAL PROPERTIES OF CELL SURFACE PROTEINS

Another exciting area where SMFS has great promise is the elucidation of the
folding and molecular elasticity of cell surface proteins. Pioneering studies
showed the ability of SMFS to stretch and manipulate bacterial membrane
proteins, thereby providing details of their unfolding pathways and of the
forces that anchor them into the membrane. These experiments were

performed on membranes that were removed from the cellular environment
which controls the protein assembly and functional state. Consequently,
studying the molecular elasticity of proteins in living cells remains very
challenging.

37-39

15.3.1 Unfolding Adhesion Proteins on Living Yeast Cells

SMFS was recently used to unfold single agglutinin-like sequence (Als)
cell adhesion proteins from . 40 Als proteins possess four

functional regions ( Fig. 15.6a ), i.e., an N-terminal immunoglobulin (Ig)-like
region, which initiates cell adhesion, followed by a threonine-rich region (T),
a tandem repeat (TR) region that participates in cell-cell aggregation and a
stalk region projecting the molecule away from the cell surface. Soluble Als
fragments containing six TR domains were attached on gold surfaces and
picked up by their terminal Ig domain using an AFM tip ( Fig. 15.6b ). Force-
extension curves showed sawtooth patterns with well-deined force peaks,
each peak corresponding to the force-induced unfolding of an individual TR
domain ( Fig. 15.6c ) . Force peaks were well described by the WLC model,
supporting further the interpretation of the sawtooth pattern. For the irst

Candida albicans