Combining steady state and temperature jump IR spectroscopy to investigate the allosteric effects of ligand binding to dsDNA

Changes in the structural dynamics of double stranded (ds)DNA upon ligand binding have been linked to the mechanism of allostery without conformational change, but direct experimental evidence remains elusive. To address this, a combination of steady state infrared (IR) absorption spectroscopy and ultrafast temperature jump IR absorption measurements has been used to quantify the extent of fast (∼100 ns) fluctuations in (ds)DNA·Hoechst 33258 complexes at a range of temperatures. Exploiting the direct link between vibrational band intensities and base stacking shows that the absolute magnitude of the change in absorbance caused by fast structural fluctuations following the temperature jump is only weakly dependent on the starting temperature of the sample. The observed fast dynamics are some two orders of magnitude faster than strand separation and associated with all points along the 10-base pair duplex d(GCATATATCC). Binding the Hoechst 33258 ligand causes a small but consistent reduction in the extent of these fast fluctuations of base pairs located outside of the ligand binding region. These observations point to a ligand-induced reduction in the flexibility of the dsDNA near the binding site, consistent with an estimated allosteric propagation length of 15 Å, about 5 base pairs, which agrees well with both molecular simulation and coarse-grained statistical mechanics models of allostery leading to cooperative ligand binding

Coarse-grained models of biomolecule dynamics and allostery

Recently, it has become increasingly accepted that thermal fluctuations take active part in functional tasks of biological molecules. We employ a set of coarse-grained models to investigate the mechanism of transmission of allosteric signal via spatial fluctuations. Our models are coarser than those in computational techniques established in molecular biology, but allow for both the identification of candidates for the essential physical structures and also the analytical determination of thermodynamic quantities that define ligand binding. The models are constructed for general classes of macromolecules and are validated through parameterisation from experiments and atomistic simulations. In the first part of this thesis we investigate the “dynamic allostery” in dimeric proteins composed of two identical subunits. We demonstrate that cooperative effects upon binding of two identical ligands can arise purely through modification of slow global vibrational modes of the protein. We parameterise the model on a test case, the CAP homodimer. Finally, we explain the role of local, fast vibrations in the allosteric effect and propose a general protocol for interpreting thermodynamic parameters of dynamically allosteric homodimers. The second part of this thesis considers allosteric effects in DNA, an example of nearly uniform elastic medium. The DNA is modeled as an elastic rod and substrate binding as local increase of its bending and twisting rigidity. This results in altered structure of normal modes and leads to qualitatively different type of dynamic allostery compared to that of the discrete models previously employed to study allosteric effects in proteins. Dynamic allostery in DNA is found always to be negative, due to an anti-correlated amplitude of thermal fluctuations at the binding site and around it. This allows us to draw conclusions about general design rules of allosteric molecules and highlight the controlling feature that biological molecules evolved to optimize their dynamics for their function

Substrate-Modulated Thermal Fluctuations Affect Long-Range Allosteric Signaling in Protein Homodimers: Exemplified in CAP

The role of conformational dynamics in allosteric signaling of proteins is increasingly recognized as an important and subtle aspect of this ubiquitous phenomenon. Cooperative binding is commonly observed in proteins with twofold symmetry that bind two identical ligands. We construct a coarse-grained model of an allosteric coupled dimer and show how the signal can be propagated between the distant binding sites via change in slow global vibrational modes alone. We demonstrate that modulation on substrate binding of as few as 5–10 slow modes can give rise to cooperativity observed in biological systems and that the type of cooperativity is given by change of interaction between the two monomers upon ligand binding. To illustrate the application of the model, we apply it to a challenging test case: the catabolite activator protein (CAP). CAP displays negative cooperativity upon association with two identical ligands. The conformation of CAP is not affected by the binding, but its vibrational spectrum undergoes a strong modification. Intriguingly, the first binding enhances thermal fluctuations, yet the second quenches them. We show that this counterintuitive behavior is, in fact, necessary for an optimal anticooperative system, and captured within a well-defined region of the model's parameter space. From analyzing the experimental results, we conclude that fast local modes take an active part in the allostery of CAP, coupled to the more-global slow modes. By including them into the model, we elucidate the role of the modes on different timescales. We conclude that such dynamic control of allostery in homodimers may be a general phenomenon and that our model framework can be used for extended interpretation of thermodynamic parameters in other systems

Modelling allosteric signalling in protein homodimers

Allostery in protein systems is a thermodynamic phenomenon. Allosteric response is driven by the free energy differences obtained in different binding events, which, in principle, contain contributions from both enthalpic and entropic changes. While traditional views of allostery have concentrated on structural changes induced by the binding of ligands (i.e. ‘‘enthalpically dominated’’), it is now increasingly recognised that fluctuations in structure can contribute to allosteric regulation. In some cases, where ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. In thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. One advantage of such a mechanism is the possibility for long range allosteric signalling. Here, changes to slow internal motion can be harnessed to provide signalling across long distances. This paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. Theoretical results are presented for the binding of cAMP to the catabolite activator protein (CAP) [1], where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. Results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins