Folding Properties Of Designed Proteins And Their Mutants

The concept of cooperativity of protein folding is that,under the process of folding or performing function,proteins display two unfolding/folding states.By many experiments,we have realized that the small naturally occurring proteins have the characterization of cooperative folding,which makes increasingly scientists believe that the cooperative folding mechanism existing in the stable and functional proteins is essential.In the forthcoming decades,de novo designed proteins have nature-like performance which are stable and functional with the development and improvement of theories and platforms of protein design,but without cooperativitity of folding.The biggest difference between small naturally occurring and de novo designed proteins was that the former experienced the natural selection but the later did not.Based on those,we proposed a hypothesis that the occurrence of cooperativity was related to the natural selection,which was just the appendage from the evolution pressure,not the stability and displaying function of proteins.Consequently,we conducted our project.The de novo designed proteins,E₁r26,D₁cy5_M1,xim5 and Rim5 A,were selected as initial templates,which were completed by Peng.X.,Meng W.and Qun Z.using ABCUS,a platform of protein design,to conduct experiments by the way of sequence degeneration.A selection system,which related the stability of protein with the CdCl2 resistance,was introduced for high throughput screening,which meaned that a better foldability of the proteins had a higher resistance to CdCl2.With a keeping level of CdCl2 concentration,iterative selection was applied on the CAC plates?CdCl2-Arabirose-Chloramphenicol?using a ePCR-MEGAWHOP method to create mutant libraries,analyze the mutant sequences,express mutant proteins and perform a battery of thermodynamic experiments including HSQC,thermal/chemical denaturation Circular Dichroism?CD?,Differential Scanning Calorimetry?DSC?and Difference Scanning Fluorescence?DSF?.The other variant E₁r26 108??+??had the similar HSQC spectra and denaturation curve CD with the natural protein 1R26,which was referred as the template of E₁r26 having the same tetrra structure,and had approximate Tm values using DSC and DSF nevertheless which of the variant was much lower than of the initial template E₁r26.The second and tetra structure of another variant 1003212 belong to E₁r26 were edge on the stability performing a cooperative folding.A variant of D₁cy5_M1 of all a-helix structure,100425,had a sigmoidal curves on an experiment of thermal denaturation CD.The stability of D₁cy5_Ml 100425,however,decreased significantly compared with the initial template D₁cy5_M1 by HSQC.The study of both thermodynamic and MIC?Minimal Inhibitory Concentration?experiments indicated that the foldability of the variants gradually decreased as the rounds of selection increasing,especially in the third round of selection which was significantly lower than the E₁r26.Experiencing the process of the degradation for the initial templates,the protein sequences gradually become disorder.In conclusion,the project adopted was right which achieved the process of anti-natural selection,from the order to the disorder of the protein sequences,meanwhile the protein sequences passed by a region where the variants would be relatively stable and cooperative on folding.Beyond the region,the stability of the variants became worse and remained a cooperative folding.Given the fact,we supposed that the occurrence of protein folding cooperativitity might not be the consequence of keeping the stability and executing the function for proteins,while just the accompaniment of evolutionary pressure,which could provide a message for the protein designer that cooperativity is seen as an unessential factor for a successful protein design.