Effects Of Small Molecules On Macromolecular Conformation

Small molecules such as urea,salts and ligands can affect the hydrodynamic behavior of macromolecules expecially for biomacromolecules.Guanine-riched DNA or RNA can fold into G-quadruplex in the presence of some cations and ligands.This structure regulates the process of cell division.Moreover,some additives,such as,urea and alcohols,can denature protein.In this thesis,we used analytical ultracentrifugation-sedimentation velocity(AUC-SV)and differential scanning calorimetry(DSC)to study the effects of some small molecules on the macromolecular conformation in aqueous solutions.The main conclusions obtained in this thesis are listed as follows:1.In the presence of some monovalent cations,human telomeric DNA segment(Tel22)can fold into a special structure called G-quadruplex.These cations can interact with phosphate groups and O6 oxygen atom of guanines by non-specific interactions and specific interactions,respectively.The results show that the standard sedimentation coefficient(s20,w)of Tel22 starts to increase when the concentration of the potassium ions(Ck+)is higher than 10.0 ?M due to the gradual formation of G-quadruplex through specific interactions.Whereas,for non-specific interaction,a higher Ck+ of 1.0 mM was needed f-or increasing s20,w.Moreover,potassium ions could promote the formation of G-quadruplex much easier than lithium,sodium and cesium ions,presumably due to its proper size in dehydrated state and the easier dehydration.Furthermore,the structural changes in this study are intramolecular change because that the molar mass of DNA at different cation concentrations was nearly a constant and close to the theoretical value of molar mass of monomeric Tel22.2.TMPyP4 as a ligand of G-quadruplex has attracted much attention.Although many methods have been used to study the interactions between TMPyP4 and G-quadruplex,many issues,such as binding number and the corresponding structural change,are still controversial.Our results show that,in the presence of Na+,the binding number of TMPyP4 per Tel22 determined by AUC-SV increases with the concentration of TMPyP4(CTMPyp4).Nevertheless,it decreases with the concentration of NaCl(CNaCl)due to the competitions between Na+ and TMPyP4.Moreover,TMPyP4 can induce the formation of dimeric G-quadruplex-TMPyP4 complex through medium affinity binding mode.High affinity binding modes have no contribution to the formation of the dimers,and the weak electrostatic binding of TMPyP4 to Tel22 has a negative effect on the formation of dimers.3.Poly(N-isopropylacrylamide)(PNIPAM)changcs its conformation from coil to globule when the temperature is above its lower critical solution tempeture(LCST,32 ?).Urea and its derivatives can affect phase transition of PNIPAM.The results show that the interactions between urea and hydrophilic groups of PNIPAM increase the enthalpy change during heating process and the interactions between urea and hydrophobic groups decrease the enthalpy changes.Moreover,the study of methylated urea reveals that the enthalpy change increases with the number of DSC cycles with the addition of osmolytes with N-H groups,presumably due to the gradual formation of hydrogen bonds with dehydrated C=O groups of PNIPAM at high temperature.These results are important and useful for us to understand the mechanism of protein denaturation.4.We have extracted the glycinin(11S)from defatted soybean flour by acid precipitation method.The results of AUC-SV reveal that 2-mercaptoethanol can decrease the amounts of the aggregates with sedimentation coefficient larger than 11 S in buffer solutions.ConA-Sepherose 4B was further used to purify 11S protein from?-conglycinin globulins.The purified 11S also has several components with different sedimentation coefficients,such as 3 S,7 S,11 S and 15 S.The change of the concentration of NaCl has no obvious effect on the relative content of each component,but it may have influence on the structure of 11S.Moreover,11S can separate to its half-molecule at low pH(pH = 3).