Thermal coefficients of methyl groups within ubiquitin and metabolic coupling of NAA and lactate in cortical neurons.

Protein folding and molecular recognition are physiological processes that are intricately linked to their dynamic signature and reflected in their thermal coefficients. Additionally, the local conformational entropy is directly related to the degrees of freedom, which each residue possesses within its conformational space. Therefore, the temperature dependence of the local conformational entropy may provide insight into understanding how local dynamics may affect the stability of proteins. Here, we analyze the temperature dependence of internal methyl group dynamics derived from the cross-correlated relaxation between dipolar couplings of two CH bonds within ubiquitin (Sabo et al Prot. Sci. 2012, 21, 562). Spanning a temperature range from 275 K to 308 K, internal methyl group dynamics tend to increase with increasing temperature, which translates to a general increase in local conformational entropy. With this data measured over multiple temperatures, the thermal coefficient of the methyl group order parameter, the characteristic thermal coefficient, and the local heat capacity were obtained. By analyzing the distribution of methyl group thermal coefficients within ubiquitin, we found that the N-terminal region has relatively high thermostability. These results indicate that methyl groups contribute quite appreciably to the total heat capacity of ubiquitin through the regulation of local conformational entropy. We have also investigated the influence of denaturant using guanidinium chloride on these thermal coefficients. From these measurements, we found that two methyl groups, L8δ1 and L73δ1 appear to be early reporters of ubiquitin unfolding. Taken together, a per residue gauge of local protein thermodynamics, as well as local responses to the presence of denaturants, offers a powerful complement to the already well-established methods for determining global thermodynamic parameters in proteins. In the socond part we developed a method to directly detect and quantify the amount of NAA within the supernatant of rat cortical neuron cultures, which is based on a specific isotope labeling scheme and two dimensional NMR spectroscopy. As hypothesized the NAA production in the neurons is dependent on lactate being present. The experiments presented here showed an increasing level of secreted NAA in the supernatant as a function of elevated lactate concentrations in the culture medium. Different control experiments revealed that the induced osmotic pressure, ionic strength of sodium lactate or increased energy supply were not the cause to promote NAA synthesis from glucose or the release of it. The internally produced lactate as one of the products of the glycolysis pathway was not evident as the only source for the NAA synthesis. The alternative hypothesis that assumes a stimulatory effect of lactate molecules on neurons to induce the release of NAA without being involved in the NAA synthesis, were not supported by the results using treatment of unlabeled lactate on neurons. The intracellular concentration of NAA was estimated to be rather constant at different lactate treatments. This might show that neurons are trying to keep the intracellular concentration of NAA at the certain level, while the excess amount is released to the supernatant. The finding of this study is shedding light on the unknown function of NAA in the brain by interconnecting the role of lactate in the myelinated neurons to the correlation of NAA synthesis and present amount of lactate.For further investigation of NAA role as an well feeding signal from neurons, treatment of oligondendoglia with different NAA concentration is suggested to observe the lactate release profile of oligodendrocytes.