Chemical Biology Methods For Visualizing The Structure And Dynamics Of Proteins

The structure and dynamic characteristics of proteins is the important foundation for its exercise of functionality. The determination of the high temporal and spatial resolution of proteins structures and dynamics is of great significance to understand the nature of life movement, the mechanism of disease as well as the research of drugs.Among the techniques, the most significant advantage of the solution NMR and cryo-electron microscopy is suitable to observe the structure and the function of biomacromolecules in solution, and these methods tend to be closer to the real physiological conditions compared with the methods of X-ray crystal diffraction etc.Solvent paramagnetic relaxation enhancement technology (sPRE), a new paramagnetic NMR method, is well suited to characterize protein dynamics. Solvent paramagnetic ralaxation enhancement affords long range restraints due to the PRE effects. For traditional paramagnetic relaxation enhancement technique (PRE), Gd3+, Mn2+, nitroxide or other paramagnetic probes were be introduced to visualize the interaction between the paramagnetic probe and protein nuclei. sPRE experiment is actually to observe exposure level information of the protein nuclear. Besides sPRE can also obtain the transient and low population dynamics information of protein like the traditional PRE.Gd-DTPA-BMA (also called Omnicscan(?)) have been used previously in sPRE experiments for structures and dynamics determination. DTPA-BMA provides eight coordinate sites to Gd3+, with the other coordination site provided by an inner-sphere H2O. In previous sPRE experiments, we found that Gd-DTPA-BMA showed hydrogen exchange with the H-N in protein under certain conditions, resulting in the abnormal sPRE values (which is significantly different from the theoretical computation values and experimental values) which could influence the accuracy of the description of protein structure by sPRE methods.In order to solve this problem, we have developed two new cosolvent paramagnetic relaxation enhancement probes, Gd-TTHA-TMA and Gd-DTPA-BEA, for investigation of protein structures. These probes can solve the hydrogen exchange problem in the formerly applied probe Gd-DTPA-BMA, leading to more accurate description on the protein structure and dynamics.Compared with the traditional Gd-DTPA-BMA probe, the Gd-TTHA-TMA that we have developed can provide 10 coordinating subunits for paramagnetic metal, eliminating the influence of relaxation exchange. Gd-TTHA-TMA improved the accuracy of the experimental value.Through the test on different proteins, the result of sPRE has confirmed that Gd-TTHA-TMA probe can provide more reliable sPRE experimental value, which can make more accurate description on the protein structure and dynamics information.However, due to the wide variety, complex and diverse functionalities of protein in nature, one type of sPRE probe cannot solve all the problems. We found that in some cases Gd-TTHA-TMA probe will combine with the hydrophobic region of proteins, influencing the accuracy of sPRE experiments. Therefore, we developed a new sPRE probe, Gd-DTPA-BEA, as the supplement with Gd-TTHA-TMA, so as to obtain more accurate and reliable sPRE information of protein structure dynamics.Afterwards, we have developed a novel methods of sPRE experiments combined with molecular dynamics simulation to analyze the protein structure and dynamics. We use adenosine kinase (AdK) protein as the model, Using the new sPRE probe that we have developed to measure the sPRE value of both the holo state and apo state of AdK protein, and then selected the fit the suitable structure of calculated value in the structure pool of the molecular dynamics simulation to visualize the structures and dynamics of AdK protein under various states. Compared with the traditional method of PRE, the advantage of the new method is that there is no need to label the proteins which could perturb the proteins stuctures and dynamics; Compared with fluorescence resonance energy transfer (FRET) techniques which can only get the information about distance between two points, the new method can obtain much more information about protein sutructures and dynamics.In addition, fluorescence resonance energy transfer (FRET) is also an important tools to study the biomacromolecular structure and dynamics. We developed two kinds of fluorescent probes, IAM-Cyanine3 and IAM-Cyanine5, to be applied in FRET experiments, which characteristics are as follows:1) compared with the formerly applied probes, we shortened the length of the linker between the luminophore and protein, making the probe have better rigidity, so as to improve the accuracy of the description for protein structure by FRET experiment; 2) we improved the fluorescent probe reaction connection method, so that it will have better reaction specificity.To sum up, this thesis mainly focused on the development of paramagnetic probes and fluorescence probes applied for the detection of protein structures and dynamics. Through the optimization of the existing structure and properties of the probes at present, enabling it to have higher accuracy in the application of the visualizing of protein structure and dynamics experiment.