Mapping the energy landscapes of supramolecular assembly by thermal hysteresis.

Understanding how biological macromolecules assemble into higher-order structures is critical to explaining their function in living organisms and engineered biomaterials. Transient, partly-structured intermediates are essential in many assembly processes and pathway selection, but are challenging to characterize. Here we present a simple thermal hysteresis method based on rapid, non-equilibrium melting and annealing measurements that maps the rate of supramolecular assembly as a function of temperature and concentration. A straightforward analysis of these surfaces provides detailed information on the natures of assembly pathways, offering temperature resolution beyond that accessible with conventional techniques. Validating the approach using a tetrameric guanine quadruplex, we obtain strikingly good agreement with previous kinetics measurements and reveal temperature-dependent changes to the assembly pathway. In an application to the recently discovered co-assembly of polydeoxyadenosine (poly(A)) and cyanuric acid, we show that fiber elongation is initiated when an unstable complex containing three poly(A) monomers acquires a fourth strand.

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions.

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.