[Keywords:] folding recognition and binding proteins, molecular chaperones of biological macromolecules
The study on the structure and function of biological macromolecules is the basis of understanding images at the molecular level. Without understanding the structure and function of biological macromolecules, there would be no molecular biology. Just like without the discovery of DNA double helix structure, there would be no central law of genetic transmission, and there would be no molecular biology today. Structural molecules can enter the complex of multi-subunits and multi-molecules from the first molecule. At the same time, with the deepening of research and the development of technical means, the life movement at the molecular level that was difficult to study in the past has gradually changed from difficult to hot. The study of protein crystallography has started from the static (time statistical) structural analysis of biological macromolecules to the dynamic (time-resolved) structural analysis and kinetic analysis. More than half of the 25 symposiums in the 13th International Biophysics Congress involved the structure and function of protein, which emphasized "dynamics", that is, the dynamic structure of protein or the relationship between its movement and molecular function, as well as its contribution to the interaction of macromolecules.
Protein folding is listed as an important topic of "2 1 century biophysics", and it is a major biological problem that has not been solved in the central principle of molecular biology. It is a very challenging job to predict the tertiary structure of protein molecule from its primary sequence, and then predict its function. The study of protein folding, especially the early folding process, that is, the folding process of newborn peptides, is the fundamental problem to comprehensively and finally clarify the central principle. In this field, new discoveries in recent years have fundamentally revised the traditional concept that nascent peptides can spontaneously fold. Among them, X-ray crystal diffraction, various spectral techniques and electron microscope techniques have played an extremely important role. At the 13 international biophysics conference, Ernst, the Nobel Prize winner, emphasized in his report that one of the main advantages of studying protein by nuclear magnetic resonance is that it can study the dynamics of protein molecules in great detail, that is, the relationship between the dynamic structure or the movement and function of the structure of protein molecules. At present, nuclear magnetic resonance technology has been able to observe the movement process of protein structure from second to picosecond in time domain, including the movement of main chain and side chain, and the folding and unfolding process of protein at various temperatures and pressures. The structural analysis of protein's macromolecules is not only to solve a specific structure, but also to pay more attention to the fluctuation and movement of the structure. For example, enzymes transporting small molecules and protein usually exist in two conformations, ligand-bound and ligand-unbound. Structural fluctuation in a conformation is a necessary prelude to conformational transformation, so it is necessary to combine spectroscopy, spectroscopy and X-ray structural analysis to study the balance of structural fluctuation, conformational change and various intermediate states formed in the process of change. For another example, in order to understand how protein is folded, we need to know the time scale and mechanism of several basic processes in the folding process, including the formation of secondary structure (helix and folding), curling, long-range interaction and complete folding of unfolded peptides. Many techniques are used to study secondary processes, such as fast nuclear magnetic resonance and fast spectroscopy (fluorescence, far ultraviolet and near ultraviolet circular dichroism).
1. A new viewpoint in the study of newborn peptide folding.
For a long time, protein folding has formed the dominant theory of self-assembly. Therefore, when studying the folding of newborn peptides, it is natural to extend the law of protein folding in vitro to the body, and use the renaturation of denatured protein as the folding model of newborn peptides. It is believed that the newly synthesized polypeptide chain in cells should be able to spontaneously fold and form its functional state without the help of other molecules and extra energy.
In 1988, Zou Chenglu clearly pointed out that the folding of new peptides began in the early stage of synthesis, not after synthesis. With the extension and folding of the peptide, the conformation is constantly adjusted. The structure formed first will affect the folding of the peptide synthesized later, and then the synthesized structure will affect the adjustment of the previously formed structure. Therefore, the structure formed in the process of peptide extension is often not necessarily the structure in the final functional protein. In this way, the formation of three-dimensional structure is a synchronous and coordinated dynamic process. In the 1990s, a class of protein with new biological functions, molecular coat proteins and, in a broader sense, helper proteins that help protein to fold were discovered, indicating that the folding of new peptides in cells usually needs help, not spontaneously.
Second, the folding of protein molecules and the role of molecular chaperones.
The three-dimensional structure of protein molecule depends on a large number of extremely complex weak secondary bonds besides * * * valence peptide bonds and disulfide bonds. Therefore, in the process of synthesis and folding, new peptides may temporarily form structures that do not exist in the final mature protein. They are often hydrophobic surfaces, which are likely to form non-functional molecules due to wrong interactions that should not exist, and even cause molecular aggregation and precipitation. According to the self-assembly theory, every step of folding is correct, sufficient and necessary. The folding process is actually a process in which the right path and the wrong path compete with each other. In order to improve the efficiency of protein biosynthesis, there should be a competitive mechanism to help the right way, and molecular chaperones came into being through evolution. Their function is to identify the wrong structures temporarily exposed during the folding process of new peptides, and combine them to form complexes, thus preventing premature interaction between these surfaces, preventing incorrect nonfunctional folding paths and inhibiting the formation of irreversible polymers, which will inevitably promote the development of folding in the right direction. From a philosophical point of view, it seems easy to refute the self-assembly theory, which violates the universal principle of contradiction. Imagine that if every step of protein's folding is correct, sufficient and necessary, wouldn't it be a great leap from quantitative change to qualitative change, from inactive peptide chain to active functional protein, which obviously violates the basic principles of philosophy? Looking at it from another angle, the process of biological evolution is full of non-directional variation, some of which are adapted to the environment and some are not. "Natural selection" eliminates the unsuitable and retains the adaptive. Isn't the folding of protein molecule similar? I think that the primary structure of protein is only the internal cause of peptide chain folding and forming the specific three-dimensional structure of functional protein. In fact, at every step of forming active protein, polypeptide chains may potentially form "incorrect" folds. Without external factors such as molecular chaperones or other helper proteins, polypeptide chains will never fold into active proteins. )
Thirdly, the mechanism of molecular chaperone.
The mechanism of molecular chaperone is actually how it recognizes, binds and dissociates the target protein. Some molecular chaperones are highly specific, such as some intramolecular molecular chaperones, and Pseudomonas esterase has its own "private molecular chaperones". It is encoded by limA gene, which is only 3 bases away from lipase gene, which may be caused by gene division during evolution. However, the general molecular chaperone recognition specificity is not high. How does it identify the object that needs its help? Now we can only say that molecular chaperones recognize unnatural conformation and ignore natural conformation. In natural molecules, hydrophobic residues are mostly located inside the molecule to form a hydrophobic core, which may be exposed after unfolding, or temporarily form a hydrophobic surface that should exist inside the molecule in the natural conformation during the folding of the nascent peptide. Therefore, it is considered that molecular chaperones are most likely to combine with hydrophobic surfaces, such as the hydrophobic side of the α -helix of Rodin molecules. But only protein with β -sheet structure can be recognized by molecular partners.
Recently, great progress has been made in the identification mechanism. Bip is a molecular chaperone in endoplasmic reticulum cavity. The specificity of Bip binding to dodecapeptide with random sequence was detected by affinity elutriation method. The results show that the motif Hy-(W/X)-Hy-X-Hy-X-Hy has the strongest binding with Bipj, and Trp, Leu and Phe are the most abundant Hys, all of which are large hydrophobic residues. Generally speaking, 2-4 hydrophobic residues are enough for binding. Another common saying is that molecular partners recognize the so-called moltenglobule structure. On the other hand, the structural analysis of the binding sites between molecular chaperones and peptides has also made some progress recently. For example, the crystal structure of PapD shows that the polypeptide binds to its β -sheet region. In GroEL, about 40kD 153-53 1 domain is the binding region of nucleotide.
The second step of molecular chaperone is to form a complex with the target protein. A very popular model holds that molecular chaperones usually form a central cavity structure in the form of aggregates. It was observed by electron microscope that fourteen GroEL molecules composed of two doughnuts and seven GroES molecules composed of one doughnut cooperated to form a hollow asymmetric cagemodel, and it was speculated that the target protein could be further folded in the middle cavity isolated from the surrounding environment without interference. Not long ago, however, a Japanese laboratory found that a subunit of GroEL, even a 50kD fragment with 78 amino acid residues removed from the N-terminal, could not be assembled into a tetrad structure, and all of them had a clear molecular chaperone function. Therefore, I think that perhaps not every part of the cyclic molecular chaperone is an effective binding site, that is, only one or several parts of the tetrad GroEL molecule composed of two layers of doughnuts can bind to hydrophobic residues or the so-called molten ball structure, and the rest plays a recognition role. Just like the detector, the whole tetrad GroEL molecule is "wrapped" in the main chain of polypeptide chain in a ring or cage structure. It moves on the chain of polypeptide chain by precession. Once the hydrophobic structure or the so-called fusion sphere structure is found in a certain recognition part of the cyclic polymer, the binding part of the polymer will combine with it to form a complex after signal transduction to inhibit incorrect folding. The above is entirely my personal guess, and I try to explain it based on the contradiction between the above two experimental phenomena. Assuming that the polypeptide chain moves in a precession way, I have no basis, but I think it should be a dynamic process, so I made some presumptuous assumptions. In addition, I think maybe X-ray diffraction can be used to detect the cage structure composed of molecular chaperones GroEL and GroES, to see whether its a×b×c is enough to accommodate a certain segment of polypeptide chain, or how its internal and external hydrophobic properties and other physical and chemical properties are, and maybe we can find support or refute the above.
These are all protein's molecular partners. Not long ago, a new term "DNAchaperones" appeared. DNA molecular chaperones bind to DNA and help it fold. In this complex, DNA molecules are surrounded on the surface of protein molecules, which is highly ordered and its structure has changed to some extent. This interaction between DNA and protein is very important for DNA transcription, replication and recombination. Or like nucleosome, DNA packaging is necessary. The structure of DNA in solution is quite rigid, and it must overcome an energy barrier before it can be transformed into the structure of its protein complex. The role of molecular chaperones is to help DNA molecules fold and distort, thus stabilizing DNA in a specific configuration suitable for protein structure. This combination is synergistic and reversibly dissociates after the complex is formed. Therefore, both DNA molecular chaperone and protein molecular chaperone are related to the interaction between DNA and protein and gene regulation. It seems that molecular chaperones are indeed closely related to the main problems that finally clarify the central principle.
Fourth, the difference between molecular chaperone and enzyme.
Unlike molecular chaperones, there are only two kinds of enzymes that help protein to fold. One is protein disulfide isomerase (PDI). The other is peptidyl proline cis-trans isomerase (PPI). Taking PDI as an example, it is well known that disulfide bonds in protein molecules are closely related to the folding of nascent peptides, and also play an important role in maintaining the structural stability and function of protein molecules. PDI is located in the endoplasmic reticulum cavity, which is rich in content and catalyzes the exchange reaction between sulfhydryl groups and disulfide bonds in protein molecules. At the same time, it is the most outstanding multifunctional protein found at present. Besides the basic function of disulfide isomerase, it is also the α subunit of proline -4- hydroxylase. It is also a small subunit of triglyceride transporter complex in microparticles and a glycosylation site binding protein. Among them, the most striking thing is that it has the ability to bind peptides with different sequences, lengths and charge distributions, and its specificity is low. It mainly interacts with the peptide backbone, but it still has a certain preference for sulfhydryl groups. According to the definition of molecular chaperone, PDI and molecular chaperone are generally considered as two different helper proteins, but China Shanghai Institute of Biophysics recently put forward a different view that protein disulfide isomerase also has the function of molecular chaperone.
The formation of natural disulfide bonds in protein molecules requires these sulfhydryl groups, which are often not adjacent to the peptide chain, to fold the peptide chain to a certain extent before they can get close to each other and form disulfide bonds correctly. The self-folding of peptide chain is a slow process, while the formation of natural disulfide bonds in protein catalyzed by protein disulfide isomerase is a fast process. On the other hand, protein disulfide isomerase has the ability to bind various peptide chains with low specificity, exists in endoplasmic reticulum at a very high concentration, is a calcium-binding protein, can be phosphorylated, and has met the requirements of molecular chaperones. Therefore, they speculate that protein disulfide isomerase may first prevent the wrong folding pathway, promote the formation of correct intermediates, and help the peptide chain to fold into the corresponding sulfhydryl pairing, thus forming the correct disulfide bond. Then catalyze sulfhydryl oxidation or isomerization of disulfide bonds to form natural disulfide bonds. They think that the enzyme activity of protein disulfide isomerase and its molecular chaperone function are not mutually exclusive, but closely related and coordinated. There should not be an absolute dividing line between molecular chaperones and enzymes that help nascent peptide chains to fold. I think that the most important characteristic of enzyme is to catalyze biochemical reaction, and the main function of molecular chaperone is to combine with the wrong conformation of new peptide, thus preventing the incorrect non-functional folding path of peptide chain and promoting its reaction in the right folding direction. Can't this be understood as indirectly catalyzing peptide chain folding? Obviously, inhibiting the incorrect folding path is equivalent to accelerating the correct reaction. Therefore, I personally agree with their views. Recent experiments provide good evidence for this hypothesis. PDI can obviously inhibit the serious polymerization of denatured glyceraldehyde-3-phosphate dehydrogenase during renaturation, and effectively improve its renaturation efficiency, which is very similar to the typical molecular chaperone GroE system in renaturation of glyceraldehyde-3-phosphate dehydrogenase.
The structure of verb (verb's abbreviation) molecular partner
At present, the only molecular chaperone that can solve the crystal structure is PapD of Escherichia coli, which helps flagellin to fold. There is also the N-terminal domain of HSP70, that is, the ATP binding domain, which also has a crystal structure. The tetragonal structure of tetramer and heptamer of GroEL has been clearly seen by electron microscope, just like two round hollow bagels stacked together. Nuclear magnetic resonance and conformational changes of various solutions are effective means to study the mechanism of molecular chaperones.
Sixth, the practical application of molecular chaperone research.
The research results of molecular chaperone will certainly deepen our understanding of life phenomena, and at the same time, it will certainly increase our ability to resist nature and survive. Because molecular chaperone plays an important role in all aspects of life activities, its mutation and damage will inevitably cause diseases, so we can expect to use the knowledge of molecular chaperone to treat the so-called "molecular chaperone disease". On the other hand, using the research results of molecular chaperones to fundamentally improve the success rate of genetic engineering and protein engineering will also play an important role in greatly improving human living standards.
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