The Use Of ¹⁹F Chemical Labeling In NMR Analysis And Protein Dynamics Characterization

Protein plays an important role in various physiological activities of the body and is the main undertaker of life activities.The dynamic characteristics of proteins determine their corresponding functions.How to obtain high-resolution structure and dynamic characteristic information of proteins,and then understand its operating mechanism,has become the focus of today's biophysics research.Temperature plays a vital role in life activities.Studying the effect of temperature on protein structure and function,especially the regulation of temperature in the intracellular environment,has always been an important challenge for researchers.In order to study the influence of temperature on the structure and function of proteins in cells,temperature sensors based on fluorescent proteins and fluorescent organics have been developed to characterize the rise and fall of temperature by sensing changes in fluorescence intensity.However,for fluorescent proteins,their intracellular components are complex and backgrounds are diverse,and it is difficult to accurately characterize temperature changes through fluorescence intensity;for fluorescent organics,the complex intracellular environment is likely to induce side reactions and lead to fluorescence quenching or enhancement;In addition,fluorescent organics are likely to affect the structure or function of proteins,all in all,temperature sensors using fluorescent proteins and fluorescent organics still face many challenges.At present,the most commonly used method for studying the dynamic structure of proteins is mainly nuclear magnetic resonance technology.Nuclear magnetic resonance refers to the process in which the magnetic spin nucleus is subjected to a magnetic field to split the Zeeman energy level and resonantly absorb radio frequency radiation of a specific frequency.Nuclear magnetic resonance is a method of spectroscopy.The protein has a spatial structure,thence two amino acids that are far apart in the sequence may interact in space.The NMR spectrum peaks of the magnetic core contained in the amino acid will generate relevant signals.By processing these signals,we can get information about the distance between two amino acids.Under normal circumstances,a protein generally contains dozens to hundreds of amino acids,and each amino acid has its own characteristic spectral peak.Therefore,for larger proteins,the resulting spectral peaks are also hundreds of thousands.These peaks are shown in the NMR spectrum.The separation that cannot be completed in the spectrum,will produce peak overlap;and the larger the protein system,the more serious the overlap;the higher the concentration,the sharp drop in the NMR signal-to-noise ratio.As a result,it is impossible to assign amino acid signals and to know the interaction between magnetic cores.Therefore,for larger protein systems,conventional NMR methods are more restricted.Based on the above scientific questions,when studying the effect of temperature on protein structure and function,this paper developed a temperature sensor based on NMR detection.We choose phosphorylated ubiquitin(pUb)as the receptor for temperature sensing.There are two-state conformations in pUb solution,namely,the relaxed state and the retracted state.Through research,we found that its two-state conformation can be regulated by temperature.We modified the cysteine(derived from point mutation)on ubiquitin through BTFA,and then introduced 19F as a probe.19F is widely used in protein structure research due to its high sensitivity,no background interference and wide chemical shift distribution.By comparing the sites,we selected K48 for mutation and introduced 19F probe,changed the temperature and collected a one-dimensional fluorine spectrum,and found that the two-state conformation of pUb changed.The ratio of the two-state conformation at different temperatures was calculated by integrating the peak area,Found a linear relationship between temperature and conformation ratio and fitted a linear equation.Through the linear equation,we only need to calculate the ratio of the two-state conformation of pUb in the sample to get the temperature of the sample at this time,and realize the development of a temperature sensor based on NMR detection.We are also based on 19F NMR to solve the research limitations of larger system proteins.We use BTFA modification to study the interaction between RSV CA domains.RSV CA is a two-domain protein with approximately 240 amino acids.It has oligomerization in solution.The introduction of 19F probe on the one hand solves the problem of peak overlap of RSV CA;on the other hand,because BTFA contains-CF3 group,it can still get high signal-to-noise ratio NMR spectrum even under the condition of very low sample concentration.We combined the PCS(pseudo-contact chemical shift)of 19F to study the interaction of the two domains of RSV CA under different conditions(crowding,self-crowding and pH),and perceive the interaction between the two domains through the shift of the 19F peak The relative position changes,it is found that BSA crowding and pH changes will affect the interaction between the two domains of RSV CA.In summary,based on a 19F chemical labeling method,this paper has developed a temperature sensor based on nuclear magnetic detection.The temperature sensor has no damage to the sample and can detect a wide temperature range(ubiquitin can withstand a temperature of more than 70? High temperature without deterioration).Since there is no 19F background in the cell,the temperature sensor is expected to be used in the cell;this thesis combines the 19F and PCS technology to study the interaction between the two domains of the dual-domain protein RSV CA,and finds that crowding and pH can regulate its interaction.The change of the relative position between the domains of a multi-domain protein is a concrete manifestation of its polymorphic conformation.The research in this thesis lays the foundation for the analysis of the polymorphic structure of RSV CA.The polymorphic conformation of RSV CA is closely related to the maturation of the capsid protein,which is essential for elucidating the mechanism of retroviral capsid protein assembly.