The Ca-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Ca exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca and several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tb is commonly used as a luminescent Ca analog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Ca and the lanthanides in CaM. Although functional assays indicate that Tb fully activates many Ca-dependent proteins, our FTIR spectra indicate that Tb, La, and Lu disrupt the bidentate coordination geometry characteristic of the CaM binding sites' strongly conserved position 12 glutamate residue. The 2D IR spectra indicate that, relative to the Ca-bound form, lanthanide-bound CaM exhibits greater conformational flexibility and larger structural fluctuations within its binding sites. Time-dependent 2D IR lineshapes indicate that binding sites in Ca-CaM occupy well-defined configurations, whereas binding sites in lanthanide-bound-CaM are more disordered. Overall, the results show that binding to lanthanide ions significantly alters the conformation and dynamics of CaM's binding sites.

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.

No abstract

No abstract

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.