Anna J. Simon

Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors

Significance Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is crucial to many cellular processes and likely could be of value in many biotechnologies. In nature, this control is often achieved using “Hill-type” allosteric cooperativity, an elegant mechanism that has, unfortunately, hitherto proven difficult to achieve via generalizable design strategies. In response, we demonstrate here a quantitative and apparently versatile means of rationally introducing this useful mechanism into a range of normally noncooperative receptors. We achieve in the best of our examples cooperativity, and thus sensitivity, experimentally indistinguishable from the theoretically expected maximum. Control over the sensitivity with which biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. Such control could likewise be of utility in artificial biotechnologies, such as biosensors, genetic logic gates, and “smart” materials, in which highly responsive behavior is of value. In nature, the control of molecular responsiveness is often achieved using “Hill-type” cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. Here, we use an intrinsic-disorder–based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. To do so, we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. The first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a preformed site. Using this approach, we have rationally introduced cooperativity into three unrelated DNA aptamers, achieving in the best of these a Hill coefficient experimentally indistinguishable from the theoretically expected maximum. The extent of cooperativity and thus the steepness of the binding transition are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy.

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.

Engineered yeast for enhanced CO2 mineralization.

In this work, a biologically catalyzed CO2 mineralization process for the capture of CO2 from point sources was designed, constructed at a laboratory scale, and, using standard chemical process scale-up protocols, was modeled and evaluated at an industrial scale. A yeast display system in Saccharomyces cerevisae was used to screen several carbonic anhydrase isoforms and mineralization peptides for their impact on CO2 hydration, CaCO3 mineralization, and particle settling rate. Enhanced rates for each of these steps in the CaCO3 mineralization process were confirmed using quantitative techniques in lab-scale measurements. The effect of these enhanced rates on the CO2 capture cost in an industrial scale CO2 mineralization process using coal fly ash as the CaO source was evaluated. The model predicts a process using bCA2- yeast and fly ash is ~10% more cost effective per ton of CO2 captured than a process with no biological molecules, a savings not realized by wild-type yeast and high-temperature stable recombinant CA2 alone or in combination. The levelized cost of electricity for a power plant using this process was calculated and scenarios in which this process compares favorably to CO2 capture by MEA absorption process are presented.

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.

Simultaneous Measurement of the Dissolution Kinetics of Responsive DNA Hydrogels at Multiple Length Scales

Recent years have seen increasing study of stimulus-responsive hydrogels constructed from aptamer-connected DNA building blocks. Presumably due to a lack of simple, quantitative tools with which to measure gel responsiveness, however, the literature describing these materials is largely qualitative. In response, we demonstrate here simple, time-resolved, multiscale methods for measuring the response kinetics of these materials. Specifically, by employing trace amounts of fluorophore-quencher labeled cross-linkers and the rheology of entrapped fluorescent particles, we simultaneously measure dissolution at molecular, hundred-nanometer, and hundred-micron length-scales. For our test-bed system, an adenine-responsive hydrogel, we find biphasic response kinetics dependent on both effector concentration and depth within the gel and a dissolution pattern uniform at scales longer than a few times the monomer-monomer distance. Likewise, we find that, in agreement with theoretical predictions, dissolution kinetics over the hundred nanometer length scale exhibit a power-law-like dependence on the fraction of disrupted cross-links before a distinct crossover from solid-like to liquid-like behavior.

Real time in vitro regulation of DNA methylation using a 5-fluorouracil conjugated DNA-based stimuli-responsive platform.

DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and many methylases have been regarded as promising targets for antimicrobial drugs. In this work, we report a stimulus responsive, self-regulating anticancer drug release platform, comprising a multifunctional DNA that upon methylation by methyltransferase (MTase) releases 5-fluorouracil (5-Fu) and in turn inhibits subsequent expression of MTase. The multifunctional DNA with anticancer drug are first methylated by DNA adenine methylation (DAM) methyltransferase (MTase) and then cut by the methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the functional DNA by the methylation/cleavage process will release the anticancer drug, resulting in inhibition of the activity of DAM in turn. Consequently, the enzyme activity of DAM MTase can be self-regulated. Furthermore, we found that the inhibition efficiency of 5-Fu significantly increase as it is functionalized with DNA.