Sixty years of protein structure analysis

At the beginning of last century, scientists thought that protein is the genetic material of life, and has a unique role With this theory being falsified, the structure of DNA, the real genetic material, has been given great attention However, as an important macromolecule of life, the importance of protein has never been ignored, and since the 1950s, scientists have been exploring the correlation between DNA sequence and protein sequence At the same time, protein sequencing and structural analysis efforts began to pay off slowly More biochemical studies reveal the importance of protein function, so the analysis of protein three-dimensional structure plays a decisive role in understanding protein function and physiological phenomena In this paper, the major historical events of protein structure analysis are reviewed, and the common methods and directions of protein structure analysis are summarized By understanding the structure of proteins, we can better understand the physical and chemical properties of biological proteins, as well as their related chemical reaction pathways and mechanisms, which play a key role in our understanding of the biological world and in the research and development of therapeutic methods and drugs At the upcoming 2015 high resolution imaging and biomedical application seminar, experts and scholars will further discuss relevant issues Major events in protein structure analysis in the past 60 years In 1958, British scientists John Kendrew and Max Perutz first published the three-dimensional structure of myoglobin with high resolution obtained by X-ray diffraction, and then the more complex hemoglobin hemoglobin So the two scientists shared the 1962 Nobel Prize in chemistry In fact, the work began as early as 1937 Then in 1960's, the protein structure analysis method was improved continuously, and higher resolution was obtained During this period, the relationship between protein sequence and DNA sequence was also found, and the central rule was proposed by Francis Crick, then the scientific community witnessed the rise of molecular biology The name of molecular biology was widely accepted and used in 1962, and gradually evolved into some branches, such as structural biology Then in 1964, Aaron Klug proposed a new method based on the principle of X-ray diffraction, which can analyze the structure of larger protein or protein nucleic acid complex Because of this research, he won the 1982 Nobel Prize in chemistry In 1969, Benno P Schoenborn proposed that neutron scattering and nuclear scattering can be used to determine the fixed position of hydrogen atom coordinates in macromolecules In the 1970s, many new methods began to develop Protein Data Bank (1971), which stores the three-dimensional structure of protein, began to appear, which is of great significance for the standardization and accumulation of protein data In 1975, a new instrument called multi wire area detector made X-ray detection and data collection faster and more efficient The following year, Robert langrid visualized X-ray diffraction data and established a computer graphics laboratory at the University of California, San Diego In the same year, Keith Hodgson and his colleagues demonstrated for the first time that X-rays from synchrotron can be used to irradiate a single crystal, and achieved good experimental results Then in 1978, NMR was first used to analyze the protein structure; in the same year, the first high-precision virus (tomato clump dwarf virus) capsid protein structure was analyzed In the 1980s, more and more protein structures were analyzed, and the description of three-dimensional structure of protein became more and more mature, and protein structure analysis was also recognized as a key step in drug development In 1983, the freeze etched structure of TMV was described under electron microscope Two years later, the German scientist John deisenhofer and others identified the bacterial photosynthetic reaction center, so they shared the 1988 Nobel Prize in chemistry The next year, two research groups analyzed the structure of protease related to HIV and replication, providing a theoretical basis for drug development for HIV In the next decade, due to the use of a large number of synchrotron assisted X-ray diffraction, thousands of protein structures have been resolved, ushering in the dawn of proteomics In 1990, the method of multi wavelength anomalous scattering (MAD) was used in X-ray diffraction crystal imaging Together with synchrotron radiation, mad has become the most commonly used method in the past 20 years Rod MacKinnon published the first high-precision potassium channel protein structure in 199, which played an important role in deepening the understanding of neuroscience Therefore, he shared the 2003 Nobel Prize in chemistry In 1999, a team led by ADA yonath and others first analyzed ribosome structure (a huge RNA protein complex) In the new millennium, more technical details have been added to the field of protein analysis In 2001, Roger Kornberg and his colleagues described the three-dimensional structure of the first high-precision RNA polymerase, so they shared the Nobel Prize in chemistry five years later In 2007, the first G-protein-coupled receptor structure analysis brought new hope for drug research In recent years, more and more large protein structures have been resolved Cryo EM ultra-low temperature electron microscopy imaging has been widely used in the study of super large protein structure imaging Common experimental methods of protein structure analysis 1 X-ray diffraction crystallography imaging X-ray diffraction crystallography is one of the earliest experimental methods for structural analysis X-ray is a kind of electromagnetic wave with high energy and short wavelength (essentially a photon beam), which was discovered by German scientist roentgen, so it is also called roentgen ray Both theory and experiment have proved that when X-ray strikes the molecular crystal particles, X-ray will produce diffraction effect By collecting these diffraction signals, we can know the distribution of electron density in the crystal, and then get the position information of particles according to the analysis Using this characteristic, Bragg father and son developed X-ray spectrometer and determined the structure of some salt crystals and diamond The first analysis of DNA structure was obtained by X-ray diffraction crystallography Later, the technology of obtaining X-ray sources has been improved, and now more X-ray sources of synchrotron radiation are used The X-ray source from synchrotron radiation can adjust the wavelength and high brightness of the ray Combined with the multi wavelength anomalous scattering technology, the crystal structure data with higher accuracy can be obtained, which has become the mainstream X-ray crystal imaging method The structure analyzed by X-ray diffraction crystallography accounts for 88% in RCSB protein data bank Although X-ray diffraction imaging has made great progress, it still has some shortcomings X-ray has a great damage to crystal samples, so low temperature liquid nitrogen environment is often used to protect biological macromolecular crystals, but in this case, the crystal surrounding environment is very bad, which may have adverse effects on the crystal Moreover, X-ray diffraction cannot be used to analyze large proteins 2 NMR MRI Nuclear magnetic resonance (NMR) was first described by Isidor Rabi (1946 Nobel Prize) in 1938 It has made great progress in the second half of the last century The basic theory is that under the influence of external magnetic field, the nucleus with lone pair electrons (the number of self selected quantum is 1) will lead to Zeeman splitting of the energy level of the nucleus, absorb and release electromagnetic radiation, that is, generate resonance spectrum The frequency of the resonance electromagnetic radiation is proportional to the intensity of the magnetic field By analyzing the electromagnetic radiation of specific atoms combined with external magnetic field, it can be used in the imaging of biomacromolecules or other fields Sometimes, NMR can be combined with other experimental methods, such as liquid chromatography or mass spectrometry In RCSB protein data bank database, there are about 11000 biomolecular structures resolved by NMR, accounting for about 10% of the total It is generally believed that compared with crystal structure, NMR can describe the real structure of biological macromolecules in cells Moreover, NMR structure analysis can obtain the structure position of hydrogen atom However, NMR is not omnipotent, and sometimes it is difficult to obtain stable signals due to the instability of protein structure in solution Therefore, computer modeling or other methods are often used to improve the structure analysis process 3 Cryo EM ultra low temperature electron microscopy imaging Electron microscopy first appeared in 1931, and was designed to obtain high-resolution virus images The image of the sample is obtained by electron beam striking the sample to obtain the reflection of the electron The resolution of the image is related to the speed and angle of incidence of the electron beam The accelerated electron beam irradiates the specially processed sample, which shows that the electron beam is reflected, received by the detector, and imaged to obtain image information The specific method is to quickly put the sample under ultra-low temperature (liquid nitrogen environment) and fix it in a very thin ethane (or water), and put it in the sample pool, and then image it under the electron microscope After the image is obtained, by analyzing the shape of a large number of the same protein in the image at different angles, computer modeling can be carried out for many times to obtain the near atomic level accuracy (as low as 2.0A) The combination of electron microscope and computer modeling and imaging is still popular after the new century With the improvement of detector technology (CCD technology, and later high-precision electronic capture and electronic counting equipment), more information and lower noise ensure high-resolution image In recent years, cryo EM has been used to analyze many proteins (or protein complexes) with very large structures (which can not be resolved by X-ray), and achieved very good results At the same time, the single electron capture technology replaces the CCD camera equipment of photoelectric conversion imaging, reduces the noise and signal attenuation in the image, and enhances the signal The maturity and progress of computer imaging technology also give cryo-EM more room for progress However, unlike X-ray, the cyro EM method does not need protein to be crystal, and the same thing is that the low temperature environment is needed to reduce the damage of particle beam to the sample In addition to the three methods introduced, computer modeling technology is increasingly used in protein structure analysis Moreover, the new analytical structure will improve the accuracy of computer modeling In the future, we may be able to use computers to build atomic level cell models and cells on chips Protein structure is of great significance for understanding biochemical reactions of living organisms and targeted drug development It has been nearly 60 years since 1958, and protein structure analysis has developed rapidly However, in the era of such efficient and cheap DNA sequencing, protein and DNA structure analysis has not entered a really high-speed development stage, which also led to so many DNA sequence data very today, but relatively few structural data With the rapid development of genomics, proteomics, metabonomics and lipomics in the era of big data, proteomics has also received more and more attention The development of high-precision and efficient structural analysis technology has always been of great significance Protein structure analysis in the future