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Effects of Crowding on the Stability of a Surface-Tethered Biopolymer: An Experimental Study of Folding in a Highly Crowded Regime
The high packing densities and fixed
geometries with which biomolecules
can be attached to macroscopic surfaces suggest that crowding effects
may be particularly significant under these often densely packed conditions.
Exploring this question experimentally, we report here the effects
of crowding on the stability of a simple, surface-attached DNA stem-loop.
We find that crowding by densely packed, folded biomolecules destabilizes
our test-bed biomolecule by ā¼2 kJ/mol relative to the dilute
(noninteracting) regime, an effect that presumably occurs due to steric
and electrostatic repulsion arising from compact neighbors. Crowding
by a dense brush of unfolded biomolecules, in contrast, enhances its
stability by ā¼6 kJ/mol, presumably due to excluded volume and
electrostatic effects that reduce the entropy of the unfolded state.
Finally, crowding by like copies of the same biomolecule produces
a significantly broader unfolding transition, likely because, under
these circumstances, the stabilizing effects of crowding by unfolded
molecules increase (and the destabilizing effects of neighboring folded
molecules decrease) as more and more neighbors unfold. The crowding
of surface-attached biomolecules may thus be a richer, more complex
phenomenon than that seen in homogeneous solution
Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions
Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (ā¼6 M) to low (ā¼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant
Entropic and Electrostatic Effects on the Folding Free Energy of a Surface-Attached Biomolecule: An Experimental and Theoretical Study
Surface-tethered biomolecules play key roles in many
biological
processes and biotechnologies. However, while the physical consequences
of such surface attachment have seen significant theoretical study,
to date this issue has seen relatively little experimental investigation.
In response we present here a quantitative experimental and theoretical
study of the extent to which attachment to a chargedīøbut otherwise
apparently inertīøsurface alters the folding free energy of
a simple biomolecule. Specifically, we have measured the folding free
energy of a DNA stem loop both in solution and when site-specifically
attached to a negatively charged, hydroxylalkane-coated gold surface.
We find that whereas surface attachment is destabilizing at low ionic
strength, it becomes stabilizing at ionic strengths above ā¼130
mM. This behavior presumably reflects two competing mechanisms: excluded
volume effects, which stabilize the folded conformation by reducing
the entropy of the unfolded state, and electrostatics, which, at lower
ionic strengths, destabilizes the more compact folded state via repulsion
from the negatively charged surface. To test this hypothesis, we have
employed existing theories of the electrostatics of surface-bound
polyelectrolytes and the entropy of surface-bound polymers to model
both effects. Despite lacking any fitted parameters, these theoretical
models quantitatively fit our experimental results, suggesting that,
for this system, current knowledge of both surface electrostatics
and excluded volume effects is reasonably complete and accurate
Experimental Measurement of Surface Charge Effects on the Stability of a Surface-Bound Biopolymer
Quantitative
experimental studies of the thermodynamics with which
biopolymers interact with specific surfaces remain quite limited.
In response, here we describe experimental and theoretical studies
of the change in folding free energy that occurs when a simple biopolymer,
a DNA stem-loop, is site-specifically attached to a range of chemically
distinct surfaces generated via self-assembled monolayer formation
on a gold electrode. Not surprisingly, the extent to which surface
attachment alters the biopolymerās folding free energy depends
strongly on the charge of the surface, with increasingly negatively
charged surfaces leading to increased destabilization. A simple model
that considers only the excluded volume and electrostatic repulsion
generated by the surface and models the ionic environment above the
surface as a continuum quantitatively recovers the observed free energy
change associated with attachment to weakly charged negative surfaces.
For more strongly charged negative surfaces a model taking into account
the discrete size of the involved ions is required. Our studies thus
highlight the important role that electrostatics can play in the physics
of surfaceābiomolecule interactions