Vibrational Relaxation in EDTA Is Ion-Dependent

Ion binding by carboxylate groups is common in biomolecules such as metalloproteins, but dynamical aspects of ion binding are not fully understood. We present ultrafast spectroscopic measurements of vibrational relaxation in the ion-coordinating carboxylate groups of EDTA, which we use as a model of carboxylate-mediated ion binding, as EDTA binds a series of divalent and trivalent metal ions with high affinity. The measurements are interpreted using a Redfield-based anharmonic model of vibrational relaxation that rationalizes trends in vibrational lifetimes in terms of vibrational energy transfer between EDTA’s asymmetric carboxylate stretching vibrational modes and lower-lying modes. Results show ion-dependent changes in complex structure and dynamics well outside the temporal and spatial resolution of common structural methods and demonstrate how vibrational relaxation measurements may contribute to exploration of ion-binding dynamics on ultrashort length and time scales

Crowding Stabilizes DMSO–Water Hydrogen-Bonding Interactions

Up to 40% of intracellular water is confined due to the dense packing of macromolecules, ions, and osmolytes. Despite the large body of work concerning the effect of additives on the biomolecular structure and stability, the role of crowding and heterogeneity in these interactions is not well understood. Here, infrared spectroscopy and molecular dynamics simulations are used to describe the mechanisms by which crowding modulates hydrogen bonding interactions between water and dimethyl sulfoxide (DMSO). Specifically, we use formamide and dimethylformamide (DMF) as molecular crowders and show that the SO hydrogen bond populations in aqueous mixtures are increased by both amides. These additives increase the amount of water within the DMSO first solvation shell through two mechanisms: (a) directly stabilizing water–DMSO hydrogen bonds; (b) increasing water exposure by destabilizing DMSO–DMSO self-interactions. Further, we quantified the hydrogen bond enthalpies between the different components: DMSO–water (61 kJ/mol) > DMSO–formamide (32 kJ/mol) > water–water (23 kJ/mol) ≫ formamide–water (4.7 kJ/mol). Spectra of carbonyl stretching vibrations show that DMSO induces the dehydration of amides as a result of strong DMSO–water interactions, which has been suggested as the main mechanism of protein destabilization

An Empirical IR Frequency Map for Ester CO Stretching Vibrations

We present an approach for parametrizing spectroscopic maps of carbonyl groups against experimental IR absorption spectra. The model correlates electric fields sampled from molecular dynamics simulations with vibrational frequencies and line shapes in different solvents. We perform an exhaustive search of parameter combinations and optimize the parameter values for the ester carbonyl stretching mode in ethyl acetate by comparing to experimental FTIR spectra of the small molecule in eight different solvents of varying polarities. Hydrogen-bonding solvents require that the peaks are fit independently for each hydrogen bond ensemble to compensate for improper sampling in molecular dynamics simulations. Spectra simulated using the optimized electrostatic map reproduce CO IR absorption spectra of ethyl acetate with a line center RMSD error of 4.9 cm^–1 over 12 different solvents whose measured line centers span a 45 cm^–1 range. In combination with molecular dynamics simulations, this spectroscopic map will be useful in interpreting spectra of ester groups in heterogeneous environments such as lipid membranes

Studying Protein–Protein Binding through T‑Jump Induced Dissociation: Transient 2D IR Spectroscopy of Insulin Dimer

Insulin homodimer associates through the coupled folding and binding of two partially disordered monomers. We aim to understand this dynamics by observing insulin dimer dissociation initiated with a nanosecond temperature jump using transient two-dimensional infrared spectroscopy (2D IR) of amide I vibrations. With the help of equilibrium FTIR and 2D IR spectra, and through a systematic study of the dependence of dissociation kinetics on temperature and insulin concentration, we are able to decompose and analyze the spectral evolution associated with different secondary structures. We find that the dissociation under all conditions is characterized by two processes whose influence on the kinetics varies with temperature: the unfolding of the β sheet at the dimer interface observed as exponential kinetics between 250 and 1000 μs and nonexponential kinetics between 5 and 150 μs that we attribute to monomer disordering. Microscopic reversibility arguments lead us to conclude that dimer association requires significant conformational changes within the monomer in concert with the folding of the interfacial β sheet. While our data indicates a more complex kinetics, we apply a two-state model to the β-sheet unfolding kinetics to extract thermodynamic parameters and kinetic rate constants. The association rate constant, <i>k</i>_a (23 °C) = 8.8 × 10⁵ M^–1 s^–1 (pH 0, 20% EtOD), is approximately 3 orders of magnitude slower than the calculated diffusion limited association rate, which is explained by the significant destabilizing effect of ethanol on the dimer state and the highly positive charge of the monomers at this pH