Ultrafast time-resolved infrared spectroscopy employing nanosecond temperature-jump initiation has been used to study the melting of double-stranded (ds)DNA oligomers in the presence and absence of minor groovebinding ligand Hoechst 33258. Ligand binding to ds(5′-GCAAATTTCC-3′), which binds Hoechst 33258 in the central A-tract region with nanomolar affinity, causes a dramatic increase in the timescales for strand melting from 30 to ∼250 μs. Ligand binding also suppresses premelting disruption of the dsDNA structure, which takes place on 100 ns timescales and includes end-fraying. In contrast, ligand binding to the ds(5′-GCATATATCC-3′) sequence, which exhibits an order of magnitude lower affinity for Hoechst 33258 than the A-tract motif, leads to an increase by only a factor of 5 in melting timescales and reduced suppression of premelting sequence perturbation and end-fraying. These results demonstrate a dynamic impact of the minor groove ligand on the dsDNA structure that correlates with binding strength and thermodynamic stabilization of the duplex. Moreover, the ability of the ligand to influence base pairs distant from the binding site has potential implications for allosteric communication mechanisms in dsDNA.

Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally stable Ca-bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations, while Ca uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

Time-resolved temperature-jump infrared absorption spectroscopy at a 0.5 to 1 kHz repetition rate is presented. A 1 kHz neodymium-doped yttrium aluminum garnet (Nd:YAG) laser pumping an optical parametric oscillator provided >70 µJ, 3.75 µm pump pulses, which delivered a temperature jump via excitation of the O–D stretch of a D2O solution. A 10 kHz train of mid-infrared probe pulses was used to monitor spectral changes following the temperature jump. Calibration with trifluoroacetic acid solution showed that a temperature jump of 10 K lasting for tens of microseconds was achieved, sufficient to observe fast processes in functionally relevant biomolecular mechanisms. Modeling of heating profiles across ≤10 µm path length cells and subsequent cooling dynamics are used to describe the initial <100 ns cooling at the window surface and subsequent, >10 µs cooling dynamics of the bulk solution.