The Origin Of The Protein Experimental Verification Of Small Molecules Induced/choice Model

“How did life begin?” is one of three central topics in the natural science field whichrequired to be answered. Since protein is not only an important biomacromolecule, but alsothe performer of vitual movement. So, origin of proteins is one of the central topics in thestudy of origin of life. Although there is no fossile of primitive protein left, we can infer thecharacteristic of ancient protein with the help of “molecular fossile” accompanied by rapiddevelopment of all kinds of “omics”. Through analyzing the distribution patterns ofsmall-molecule ligands in protein space, we proposed that the primitive protein architectureswere generated by the induction/selection of small molecules. In this thesis, this hypothesiswas preliminarily valiadated by the study of characteristic and structure of ancient proteinconstructed by primitive amino acids, and the study of cofactors induced effects on theconformation transfer of random peptides consisting of primitive amino acids set.Objectives: To elucidate whether an ancient redox protein can be construsted by aprimitive amino acids set? Secondary structure characteristic of random polypeptidesconsisting of4kinds of primitive amino acids, secondary structure characteristic of randompolypeptides consisting of15kinds of amino acids which encode by VNM, and whethercofactors can induce transfer of the conformation of random polypeptides?Methods: Through substituting “Late amino acids” by “Early amino acids” of aflavodoxin by rational design, mutant flavodoxin recombinant vector was constructed and itscharacteristic properties were evaluated.（Val^x/Glu^y/Lys^z/Ser^Z） random co-polypeptides wassynthesized by N-carboxyanhydride open-ring reaction, the secondary structure characteristicof random co-polypeptides and the effect of organic cofactor on the conformation transitionof random co-polypeptides were determined by Circular Dichroism spectrum analysis. ADNA library encoding random polypeptides consisting of15kinds of primitive amino acidswas constructed by strategy of library in small cassette, consequentlyly a random peptideslibrary was obtained by in vitro translation. The secondary structure characteristic andhydrophobic domain of random polypeptides were determined by Circular Dichroismspectrum analysis bis-ANS binding assay. The effects of cofactors on the conformationtransition of random co-polypeptides were invested by Circular Dichroism spectrum analysis bis-ANS binding assay also.Results: Mutant flavodoxin has similar structural characteristics to wild-type protein,although the semiquinone and hydroquinone flavodoxin mutants possess lower stability incomparison with corresponding form of wild-type flavodoxin, the redox potential of doubleelectron reduction(E_ox/hq)arrived at-360mV, indicating that flavodoxin mutant consititutedsolely by early amino acids can exert effectively electron transfer activity. The secondarystructure of （Val^x/Glu^y/Lys^z/Ser^Z） random co-polypeptides include α helix and random coil,there is no β sheet was induced by ATP, NAD and NADH. The β turn were induced by lowconcentration organic cofactors, the conformation transition of β turn, α helix and randomcoil were induced by higher concentration organic cofactors. Random polypeptides consistingof15kinds of primitive amino acids which encoded by VNM is not only soluable but alsohas a secondary structure characteristic of protein. Cofactors can induce conformationtransition of random polypeptides and the effect is different between different cofactors. Theinducing effect of ATP shows as decrease of β sheet and random coil, and the increasedcontents of α helix and β turn. The inducing effect of NADP+shows as increase of β sheetand decreased content of random coil, the contents of α helix and β turn changed slightly. Theinducing effect of NAD⁺shows as increase of β sheet and decreased content of α helix and βturn, the contents of random coil changed slightly. The inducing effect of NADH shows asa slightly increased β sheet and a slightly decreased α helix, the contents of β turn andrandom coil changed very slightly. The inducing effect of Mg²⁺shows as a slightly increasedβ sheet and a slightly decreased β turn, the contents of α helix and random coil changed veryslightly. ATP, NAD⁺and NADH can induced the formation of hydrophobic domain ofrandom polypeptides.Significance: An ancient redox protein constructed by14kinds of primitive amino acidswas validated in this paper. Furthermore, it was finded that random polypeptides consisting ofreduced amino acids set has secondary structural characteristic of protein,15kinds ofprebiotic amino acids set is a foldable set, cofactors can induce the conformation transition ofrandom polypeptides, ATP, NAD⁺and NADH can induce the formation of hydrophobicdomain of random polypeptides. These results not only provide an experimental verificationfor “small molecular induced the origin of protein” hypothesis, but also can provide a novel solution for protein engineering.