Sequence Redesigned Ras Binding Proteins And Analysis Of Their Structure,Interaction And Stability

Protein design is an active,exciting and challenging research area.Combining the theoretical design with experiments is an important part of protein design.In this paper,we focus on the construction of efficient detection systems for designing protein-protein interactions,the application of protein design in improving thermal stability of a protein of important interaction functions,and the analysis of the structure and function of designed proteins.For the purpose of assaying protein-protein interactions of de novo designed proteins with high efficiency,high accuracy and high throughput,we implemented systems for rapid detection and directed evolution of protein-protein interactions.These systems are based on the protein fragment complementation of dihydrofolate reductase(DHFR)form mouse and Escherichia coli,respectively.The system of murine DHFR-based Protein-fragment complementation assay(PCA)is of high sensitivity,high throughput,and low false positive rate.The E.coli DHFR-based PCA system is flexible with adjustable dynamic ranges.The combination of two systems allows us to detect and distinguish of protein-protein interactions in different affinity ranges,and to fine tune protein-protein interactions through directed evolution.Using computational sequence design,we developed an approach to enhance the thermal stability of a protein while maintain its interactions.Specifically,we redesigned the Ras-binding domain of Raf(Raf-RBD)by ABACUS program(A Backbone based Amino aCid Usage Survey)with Raf-Ras interface residues retained.The binding property of selected ten designed sequences was tested by the DHFR_PCA system.All designed proteins can interact with Ras.The thermal stability of four of the designed proteins was analyzed by DSF,DSC and HSQC.The thermal stability of four proteins was much higher than that of the native protein Raf-RBD.The designed protein dRafX6 maintain high affinity with Rass.In addition,it exhibits extremely high thermal stability,with melting temperature 40? higher than the native Ras-RBD.We carried out further analysis on dRafX6.Through the NMR analysis of three-dimensional structure,NMR chemical shift perturbation of dRafX6 and Ras,we can conclude that the three-dimensional structure and interaction interface with Ras of dRafX6 are similar to those of Raf-RBD.On the basis of this,we constructed a series of intermediate mutants perturbing the sequence of Raf-RBD toward dRafX6 or vice versa,the mutation sites selected on the basis of energy calculations using the ABACUS program.The thermal stability of those mutants suggested that the significant increase in thermastability of dRafX6 relative to the original protein is probably a result of the redesigning of the overall sequence,which may not be attributed to effects of mutations at a few sites individually:perturbing the native Raf-RBD sequence by one or two mutations towards the dRafX6 sequence generally did not show siginficant effects on thermostability;on the other hand,perturbing the dRafX6 sequence in the reversed direction may result in significant decreases in thermal stability.These results suggest that the effects of overall sequence redesign can be very different from analysis of mutations at individual sites.The strategy of redesign of a protein sequence to improve its property while maintaining its functionally required regions is a general one.We illustrated the effectiveness of the ABACUS method in this approach,which can be applied to other functional protein redesign.We analyzed the reasons of high thermal stability and epistasis in stability through the detection of dRafX6 and Raf-RBD mutants.We found that the same mutations have different stability effects in different genetic backgrounds(Raf-RBD and dRafX6),mainly due to their different environments.Through the analysis of the strcutural information of Raf-RBD and dRafX6,we found that the surface electrostatic interaction,hydrophobicity of the amino acid,the hydrogen bond are the reasons for epistasis and play an important role in protein stability.