(1) Determination of protein primary structure
The determination of protein primary structure is also called protein sequence analysis. The basic method is:
①The specific cleavage of the polypeptide chain by chemical cleavage method and protease hydrolysis method;
② Determine the sequence of each purified small peptide segment one by one;
③ Determine the arrangement order of the small peptides according to the overlapping regions in the amino acid sequences of the peptides;
④ Complete the sequence analysis of the entire polypeptide chain.
Although protein sequence analysis has been automated, it is still time-consuming, complex and expensive. After the advent of recombinant DNA technology, people can directly deduce the amino acid sequence of a protein from cDNA or gene sequence, which is fast and economical, and has become the most commonly used method for determining the primary structure of proteins.
(2) Determination of protein three-dimensional structure
According to the state of the protein, the methods for determining the three-dimensional structure of the protein are divided into two categories:
①Apply X-ray crystallography and neutron diffraction to determine protein molecular conformation in crystals (Figure 14-6);
②Apply nuclear magnetic resonance (NMR), circular dichroism spectroscopy, laser Raman spectroscopy, fluorescence spectroscopy, UV-difference spectroscopy and hydrogen isotope exchange to determine protein conformation in solution.
Fundamentals of X-ray Crystal Diffraction
1. X-ray crystal diffraction pattern
The conformation of protein molecules was determined by X-ray crystallography, and the results were reliable. However, the conformation of a protein molecule in a crystal is static compared to its conformation in solution. Therefore, unstable transition state conformations cannot be determined using protein crystals. Also, many proteins are difficult to crystallize, or to obtain single crystals large enough for structural analysis. In addition, X-ray crystal diffraction has a longer workflow.
2. nuclear magnetic resonance
Nuclear magnetic resonance refers to the physical process of nuclei whose nuclear magnetic moment is not zero. Under the action of an external magnetic field, the nuclear spin energy level undergoes Zeeman splitting (Zeeman splitting), and the resonance absorbs the physical process of radio frequency (RF) radiation of a specific frequency. . In recent years, NMR methods have been successfully applied to determine the three-dimensional structure of small proteins. The NMR method does not require the preparation of protein crystals, but the method is limited to analyzing small proteins up to 150 amino acid residues in length.
The basic process of determining the three-dimensional structure of protein by NMR
Other methods can determine the local conformation of protein molecules in solution, but it is difficult to obtain the complete three-dimensional structure of protein molecules, which has great limitations in application.