Study On The Mechanical Properties Of Surface Repeat Domains Of Protein Rib In Group B Streptococcus

An important cause of pneumonia,meningitis,sepsis and other diseases in neonates is Group B Streptococcus(GBS),cell surface proteins of which can cause protective immunity.Type III strains of GBS commonly express a protein called Rib(Resistance to proteases,Immunity,Group B)with a highly repetitive tandem sequence called Rib R(Single Rib Domain).Multiple tandem Rib R form a long rod-like structure with certain rigidity,and the movement distance of bacteria can be controlled by the number of tandem Rib R.Study of structure of Rib R showed that Rib R may have evolved from another protein,Rib L(Rib Long),which is similar in structure to immunoglobulin-like proteins through domain atrophy.However,the reason why Rib L was replaced by Rib R in evolution is unclear.In this study,Rib R,Rib L and related variants were stretched at different speeds using single-molecule force spectroscopy(SMFS),which is based on atomic force microscopy(AFM).The worm-like chain(WLC)model was used to perform statistics and analysis on the unfolding force and contour length increment of the target protein,and the differences in mechanical properties of each protein were compared.The results showed that Rib R with atrophied structure had stronger mechanical properties,and the presence of more fragments in Rib L than Rib R would reduce the unfolding force of the protein.Besides,the molecular dynamics(MD)method was used to explain the unfolding process of Rib R and Rib L in terms of tensile force,number of hydrogen bonds,electrostatic force and van der Waals force,and secondary structure.Rib R without redundant fragments is more compact and has stronger steric structure stability.This study analyzes the difference between Rib R and Rib L from a mechanical point of view,and speculates that during the evolution process,Rib L discards some redundant fragments to obtain stronger mechanical properties.This discovery provides a new direction for the development of vaccines for GBS-related diseases.In addition,in the construction of the AFM stretching system,the experiment was adjusted and optimized by using the specific covalent combination of Spy Tag and Spy Catcher,a peptide-protein combination isolated from Streptococcus pyogenes.From this we learned that protein obtained by both of the prokaryotic expression system and the cell-free system can directly modify the AFM tip and substrate without traditional protein purification process.This conclusion provides greater convenience for future related research.