Herschel M. Watkins

Determinants of the Detection Limit and Specificity of Surface-Based Biosensors

Here, we employ a model electrochemical DNA sensor to demonstrate that the detection limit and specificity of surface-based sensors often are not dependent on the true affinity of the probe for its target but are simply dependent on the effective probe concentration. Under these circumstances, the observed affinity (and thus the sensors detection limit and specificity) will depend on the density with which the probes are packed on the surface of the sensor, the surface area, and even the volume of sample employed.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Significance The relationship between proteins unfolded under physiological conditions and those unfolded by chemical denaturation remains controversial. Specifically, although FRET studies suggest that unfolded proteins invariably contract with falling denaturant levels, scattering studies argue that they do not. Here, we explore the origins of this discrepancy using PEG as a negative control. Scattering indicates that, as expected, the polymer’s dimensions are denaturant-independent. The dye-labeled polymer, nevertheless, exhibits denaturant-dependent changes in measured transfer efficiency similar to those seen for unfolded proteins. This similarity raises questions regarding the interpretation of such changes as being indicative of hydrophobic or hydrogen bond-driven collapse. 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 (∼6 M) to low (∼1 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 charged-but otherwise apparently inert-surface 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 ~130 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.

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 ∼2 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 ∼6 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.

Observation of correlated X-ray scattering at atomic resolution.

Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles—such as proteins in solution—may be feasible in the future.

Angular correlations of photons from solution diffraction at a free-electron laser encode molecular structure

An atomic twinning structure is observed by averaging intensity correlations from many snapshots of gold nanoparticles in solution.

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 biopolymers 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 surface-biomolecule interactions.

Correlated scattering: probing atomic structure of molecules and nanoparticles

In 1977, Z. Kam theorized that correlations of scattering patterns, measured by exposing a solution of randomly oriented identical particles to x-ray radiation, could yield detailed information on the internal structure of the individual particles [1]. During a single exposure (whose duration should be short compared to the particle rotational diffusion time), physical correlations arise whenever multiple photons scatter from the same particle into different directions. By averaging correlations from many exposures, we have demonstrated that one can extract this correlated signal from a background of uncorrelated single-direction scattering events from different particles [2]. This additional information can be used to place constraints on model structures of the particles under investigation, providing a method of structure refinement to atomic resolution. We recently observed correlated scattering from solutions of ~10^9 silver nanoparticles exposed to synchrotron radiation at a microfocus beamline at SSRL [2]. By autoand crosscorrelating the Bragg rings 111 and 200, five correlation peaks were resolved corresponding to the structure and symmetry of silver’s reciprocal lattice. To transition from nanoparticles to biomolecule studies, we have performed several experiments at x-ray free electron laser centers (SLAC and SPring-8), and are working to refine analysis techniques.