Simona Golič Grdadolnik
Period: 1.7.2019 - 30.06.2022
In this project, we would like to study the structural and bonding properties of water near nonpolar groups in molecules where both polar and nonpolar groups are present. We plan to study the water solutions series of short alcohols and representative amides, with different ratios between the size of polar and nonpolar regions by applying the same strategy as we used it in the case of studying the impact of purely hydrophobic molecules on water structure. We expect that the vicinity of the polar groups will influence the structure and strength of bonding of water molecules near the hydrophobic part of molecules, which will manifest in differences in observed red shifts of perturbed water OH stretching for a particular solute. The measurement of infrared spectra used in our previous study will be upgraded with new ATR appliance which enables to study more diluted solutions with respect to conventional ATR cell. These types of experiments are necessary because we have to measure pure solutions free of solute aggregates. To simplified the OH stretching contour instead of observing the OH stretching of pure H O water, a small amount of D O will be added (~1.4%). The double subtraction method will be used to extract the amount of perturbed water by solute from the vibrational spectra. Extensive QM MD simulations based on recently developed DFT functional, which have been proven to properly account for dispersive forces, will be performed in parallel. Physically relevant information will be derived from the simulations and critically compared with the available experimental and theoretical data which will be used further to establish a consistent picture of the solute effect on water structure. We plan to apply the structural sensitive indicators in vibrational spectra developed in our group on an intrinsically unstructured domain (proNGF, the precursor of Nerve Growth Factor). NMR spectroscopy will be used to clarify the cellular role of proNGF, i.e. the influence of the intrinsically unstructured propeptide on mature NGF structure, and connection between the dynamic properties and predicted intrinsically unstructured nature of the protein. We will focus on investigation of the functional difference of proNGF in the interaction with its partners, by applying biochemical/biophysical and structural approaches, including vibration spectroscopy and solution NMR. These investigations we would like to address several open questions remain to fully understand the cellular role of proNGF: how is the structure and accessibility of mature NGF affected by the presence of the propeptide? Which are the dynamical properties of the pro-peptide and how could they relate with the predicted intrinsically unstructured nature? How can we explain the different interactions with the receptors based on the structure of the proteins? In addition, the strength of hydrogen bonding between the water molecules and proteins determined from the vibrational spectra will be implemented in simple algorithms to predict the protein structure in water and helix to coil transitions. Opposed to the two-state approach with the semi-empirical formulas, that results in characteristics of the whole process, like the enthalpy or heat capacity change during the process, we suggest to use formulas that allow to estimate the energies of inter- (water-polypeptide) and intramolecular H-bonds (NH...O=C). Derived formulas can be fitted to experimental data, resulting in a more detailed and well-posed description, as compared to the two-state approach with the semi-empirical formulas. The goal of improvement of modified folding/helix-coil models is to reproduce elliptical phase diagram and cold denaturation. We will check this approach through experiments on several proteins, including IUP.