Effects Of Macromolecular Crowding Environment On Alkaline Phosphatase Unfolding Process And Dipeptide Assembly

Protein folding and peptide self-assembly have been extensively studied in vitro,but these experiments are generally done in dilute solutions,which vary greatly from the real intracellular environment.Cells are tightly packed with various biological macromolecules(proteins,protein complexes,nucleic acids,ribonucleoproteins,polysaccharides,etc.).These macromolecules occupy a significant fraction(~40%)of the total cellular volume,suggesting that the intracellular environment is extremely crowded.It affects the mass transfer,diffusion and response between intracellular substances.Therefore,it is important to study the effect of macromolecule crowding on protein folding and peptide self-assembly in vitro,which is of great significance to elucidate the real behavior of intracellular biomolecules in vivo.In this paper,we investigated the effects of macromolecule crowding environment on the activity,conformation and stability of alkaline phosphatase(ALP)and the structure and morphology of Fmoc-FF self-assembly.The main research contents are as follows:1.The effects of a variety of spectroscopic techniques on the folding process of macromolecules to ALP were studied.In our experiments,we found the following conclusions.(1)Due to exclusion volume effects,the activity of ALP increases compared to the results of no crowding agents.(2)The addition of crowding agents didn't impact the three-state changes of ALP unfolding process.In the N?I process,PEG 4000 and Dextran 70 enhanced the structural stability of ALP to some extent.In the I?U process,PEG 4000,Dextran 70 and Ficoll 70 couldn't enhance the structural stability of ALP.(3)The results of intrinsic fluorescence spectra,ANS fluorescence spectra,three-dimensional fluorescence spectra and synchronous fluorescence spectra,suggested that effects on macromolecular crowding on ALP unfolding process were complex,changing with the type,size and shape of crowding agents.2.The co-assembly behavior of protamine and Fmoc-FF in dilute solution and macromolecule crowding environment was studied by using a variety of test methods.The results showed that:(1)Fmoc-FF could assemble to form aligned nanofibers in alkaline aqueous solution(pH> 9),and the assembly solution had a strong birefringence effect under an optical microscope.(2)In dilute solution,under different pH conditions the morphologies of the fibers formed after co-assembly of protamine and Fmoc-FF were different.At pH = 8,the co-assembled fibers were right helical nanobelts.At pH = 9,the assembled fibers were left helical nanobelts.(3)At pH = 9,with the mass fraction of protamine increased,the co-assembled fiber spiral reduced,and the diameter became gradually larger.(4)In macromolecular crowding environment,the dispersion of co-assembled fiber was better,and the fiber diameter became smaller.