In Aqueous Solution, And Establishment Of Protein Secondary Structure Analysis Methods Of The Infrared Camp Receptor Protein Conformation Research

The protein is ubiquitous in the life activities, not only does the movement of life and the body’s metabolism rely on a variety of proteins, but also genetic information replication, transcription and expression are dependent on the protein. The function of a protein depends on its spatial structure. Although X-ray crystallography and NMR techniques are able to obtain protein structural information, and there are more than80,000proteins’structural data in PDB (Protein Data Bank) by far, fast and simple secondary structure estimation methods are still important technique for structural biologists. At present, the main methods to estimate protein secondary structure are spectrum method, such as infrared spectroscopy, ultraviolet spectroscopy, fluorescence spectroscopy, circular dichroism and so on, which have their own strengths respectively. X-ray crystallography can provide complete protein crystal structure information, but it requires high-quality single crystal samples, which is difficult to get. NMR technique can determine the conformation of the protein in solution, but is generally limited to study the small high purity protein with the molecular weight not exceeding20kD; In addition, the whole determination process is slower. Circular dichroism spectroscopy is widely used in determination of protein and peptide conformation in solution, but only in the clear solution within a very narrow concentration range. UV and fluorescence spectra are limited to the determination of a few protein molecules containing aromatic and heterocyclic conjugated ring system of the chromophore. Fourier transform infrared spectroscopy (FTIR) technology is a powerful means to study protein secondary structure which can overcome the inadequacies of the above analysis method with high accuracy and stability. FTIR technique has extensive and in-depth study domestically and internationally. However, due to the limitations of the experimental conditions in our country, the study of protein-infrared has been difficult to carry out in aqueous solution.The cAMP receptor protein (CRP) from Escherichia Coli is a classic transcription regulator in prokaryotes. After binding the cAMP, CRP undergoes an allostery and recruits the DNApromoters, then actives the RNA polymerase to initiate transcription of many catabolite genes. Based on the studies of transcriptomics and bioinformatics, the transcriptions of nearly200different promoters are regulated by CRP. As one of the most studied transcriptional regulators, and the similar cAMP-and DNA-binding motif found in other important function protein, CRP has become an important paradigm for allostery. The crystal structures of CRP-cAMP, CRP-cAMP-DNA, and CRP-cAMP-DNA-RNAP complex have all been determined. The study on CRP using methods ranging from biochemical, biophysical techniques and molecular dynamics, all show that the cAMP binding to CRP first provokes the local perturbation at the directly contacting points, which then propagates to whole molecule and leads to global transition. Recently, the structure thought to be the last one for the understanding of allostery mechanism induced by cAMP binding, apo-CRP, was determined. Although it was truly contributed to the understanding of the mechanism, there are many issues to be studied. A precisely determined structure of apo-CRP or the CRP-cAMP complex has a wider significance.To solve the problems, we carried out our research about the methodology of protein-infrared and the allostery mechanism of CRP to form this dissertation. The conclusions are summarized as follows:1. Our laboratory newly purchased the first protein-infrared analyzer for aqueous solution, this dissertation established a drying system of protein infrared in aqueous solution for domestic experimental conditions, the usage of purge of dry air can avoid of the air interference of water vapor, which makes smooth progress of the protein-infrared experiment in aqueous solution.2. In this thesis, based on existing laboratory instruments, we established a Fourier transform infrared analysis method for protein secondary structure analysis in the aqueous solution. We regulated the experimental conditions, sample preparation methods, experimental operation and data processing, and applied to the secondary structure analysis of the soluble guanylate cyclase heme domain protein and the comparison of FTIR and circular dichroism method. In contrast, FTIR has a higher accuracy and stability.3. In this thesis, crystallization attempts were made on CRP and its several mutants. First, we have the growth of the D138L CRP mutant crystal which is easier to obtain, and then the D138L CRP microcrystalline was used to induce the crystallization of apo-CRP and other mutants, at last we get crystal of apo-CRP, L148R CRP and G53H CRP, and the G53H CRP mutant crystal data was collected.4. Because of the cAMP-binding motifs founded in other proteins, we also studied the role of loops3and4in the interdomains and intersubunits communication of CRP. Two mutants were constructed according to the differences in sequence alignment between CRP and the other cAMP-dependent proteins, namely the insertion KGSKM between E78and G79in loop4(Mutl), and then the deletion of E54-G56in loop3(Mut2). In the present study, wide-type (WT) CRP and the two mutants were digested by subtilisin and chymotrypsin, to obtain the six CRP core fragments, known as α-CRP (S-WTCRP, CH-WTCRP, S-iCRP, CH-iCRP, S-idCRP, and CH-idCRP). Together with the results of biochemical and biophysical experiments, we got these conclusions:the changes in loop3and4perturb the cAMP binding pocket and alter cAMP binding behavior. The allosteric signal transmission process is most likely the result of overall realignment of the two subunits within a CRP dimer.