Interactions between trimethylamine n-oxide and elastin-like polypeptides trimethylamine n-oxide at the air/water interface
Open Access
- Author:
- Liao, Yi-ting
- Graduate Program:
- Chemistry
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- August 05, 2015
- Committee Members:
- Paul S Cremer, Thesis Advisor/Co-Advisor
- Keywords:
- Trimethylamine N-oxide
surface tension
Sum frequency generation
protein folding
water - Abstract:
- Trimethylamine n-oxide (TMAO) is a natural osmolyte that is well-known to stabilize protein function and structure in solution. Despite its action on proteins is widely exploited, the molecular mechanism behind the action is still debatable. Protein stabilizers are proposed to stabilize protein through preferential exclusion from protein surface or by enhancing bulk water structuredness. In the first part of the thesis, we studied the stabilizing effect of TMAO on elastin-like polypeptides (ELPs). The interactions between TMAO and ELPs are studied by measuring the depression of lower critical solution temperature (LCST) of ELP by TMAO and by measuring surface tensions of TMAO solutions. The experimental results indicate there is no measurable interaction between TMAO and ELPs. However, TMAO indeed slightly accumulates at the hydrophobic surface. In addition, the influence of TMAO on the hydrogen bonding network is investigated via infrared spectroscopy. TMAO affects hydrogen bonding strength mainly by the NO dipole that serves as a hydrogen bond acceptor. Nevertheless, such influence is short-ranged and cannot affect bulk water structuredness. Molecular dynamic simulation results demonstrate that TMAO directly contacts the surface of ELP; moreover, it decreases the tetrahedral ordering of water structure both in the bulk solution and at the ELP surface. Such results contradict the central tenants of contemporary theories for the means by which TMAO can lead to protein stabilization. Namely, TMAO’s effects are clearly not due to exclusion from the protein surface or making of water structure. Instead, a new model is required that takes into account the fact that TMAO preferentially binds to the protein structures and stabilizes the collapsed state on entropic grounds. Curiously, these results stand in contrast to the mechanisms of actions of other osmolytes such as glycine, sarcosine, dimethyl glycine, and betaine, which all appear to favor protein stabilization via preferential exclusion mechanism. The intracellular environment is very crowded, and up to 70% water in a cell is present at an interface. Therefore, it is of interest to study water structure in a confined geometry. In the second part of the thesis, the effect of TMAO on the interfacial water structure at the air/water interface is studied to gain further insights into the influence of TMAO on water structure at hydrophobic portions of protein surface. Previously, our lab discovered that TMAO adopts a side-on orientation (methyl group-down) at the air/water interface. This indicates the methyl groups of TMAO are more hydrophilic than previously thought. Further studies show TMAO reorients slowly at the air/water interface during the adsorption process. In the early stage of adsorption, because the surface concentration is low, TMAO is able to adopt a side-on orientation to hydrate both the methyl groups and the NO dipole at the water-sufficient surface. In the later stage of adsorption, because the surface pressure is high, TMAO needs to adopt a surfactant-like orientation (NO dipole-down) to keep NO dipole sufficiently hydrated at the water-deficient surface. In addition, the interfacial water structure is strongly enhanced when TMAO reorients from a side-on orientation to a surfactant-like orientation. This is because the well-organized vertically-orientated NO dipole of TMAO at the interface aligns water molecules underneath. This discovery indicates the effect of TMAO on the interfacial water structure is strongly orientation dependent. In addition, the adsorption of TMAO at the air/water interface is an activated-diffusion process with an energy barrier about 22.5 kcal/mole. The adsorption energy barrier may be due to the increase in the surface pressure and the molecular realignment. The future direction for the second subject is also proposed in the final chapter of the thesis.