Experimental Analysis Of Designed Proteins And Their Directedly Evolved Mutants

The basic goal of the de novo designed proteins is about the de novo designed amino acid sequences to fold into desired structures which is a way to reach a more thorough understanding of how primary structure as amino acid sequences encode protein structures and decide the function. Notwithstanding several successful experimental validation examples, there are noteworthy limitations of the applications of computational protein design. The bottleneck of protein design is still the low design success rate caused by not deep enough understanding of protein folding, not mature protein design method. And validating the design results completely based on the structure analysis by experimental tools is high cost and phugoid, which makes the validation of the design method lacking systemic and less reliable feedback information etc and limits the improvement of the design method. Here, we establish a high efficient experimental system in which the structural stability of a protein of interest (POI) is linked to the antibiotic resistance of bacteria cells to achieve the high efficient validation of the designed proteins’foldability. We also use this system to directly evolve the poor initial foldability design amino acid sequences. The results just contains only a small design sequence errors, could be rectified by directed evolution introducing some point mutations. Finally, we solved three designed proteins’three-dimensional structures using nuclear magnetic resonance(NMR) and X-ray crystal diffraction. One of them is a directly designed protein and the other two proteins are evolved designed protein mutants. All the three proteins share the same topological structures with the designed targets. The high resolution crystal structure alignment between the designed protein mutant and the design target reveals some sequence sites may have a crucial impact for the three-dimensional structure. Then we also analysis these sites’structure effects by directed mutagenesis. We analysis the three proteins’heat denaturation process using circular dichroism (CD) and differential scanning calorimetry (DSC). The results showed these designed proteins have high thermostability, at the same time, their unfolding process lacks the cooperativity. At this point, although our designed proteins are different from most natural proteins, sharing the similar results with most reported artificial designed proteins. The reason needs further research, at least proving protein folding cooperativity is not necessary to form a correct and stable three-dimensional structure.Based on the already finished work, we select multiple highly rules spatial structures, respectively as theoretical design templates, to comprehensively identify the theoretical design success rate. Nuclear magnetic resonance spectra results show that in the first round of the design results,80% of designed proteins can fold to stable spatial structures. At the mean time, the solubility of the 50% designed proteins is poor. Based on the experimental results, we improved our design method and designed second round proteins. Nuclear magnetic resonance spectra results show that the second batch of design results not only form the stable three-dimensional structures, but the designed proteins’solubility has significantly improved.