Denaturation And Folding Of Proteins And Peptides In Aqueous Solutions And At Interfaces

Proteins play vital role in biological processes.Denaturation,absorption and folding of proteins are highly related to their corresponding functions.Study of behaviors of protein and peptides in solution and at interfaces can help to understand the influences of environmental factors on the structures and functions of proteins.In this dissertation,we studied the denaturation,adsorption,folding and other behaviors of proteins and peptides in aqueous solutions and at interfaces.We discussed the influence of denaturant,osmolyte,ions on the behaviors of proteins and peptide.The main contents are as follows:(1)The mechanism of urea-induced lysozyme denaturation and the counteraction of betaine to the denaturation was studied by low field nuclear magnetic resonance(LF-NMR)relaxation spectroscopy.The presence of lysozyme led to the merge of urea and water peaks in the spin-spin relaxation time(T₂)inversion spectrum of urea-lysozyme solution,indicating that the hydrogen exchange between urea and water happened.It reveals that urea directly binds to the surface of lysozyme and further indues protein denaturation.A betaine-water peak was observed in the T₂ inversion spectrum of urea-betaine-lysozyme solution,indicating that urea left the protein surface and the hydrogen exchange between urea and water was inhibited.It demonstrates that betaine stabilizes the structure of lysozyme by pulling urea away from the protein surface.¹H-NMR and FTIR spectra reveal that betaine associates with urea through hydrogen bonding.(2)The interactions between guanidinium ion(Gdm⁺)or ammonia ion(NH₄⁺)and amino acids are investigated by LF-NMR relaxation spectroscopy.No significant change of spin-lattice relaxation time(T₁)of Gdm Cl or NH₄Cl solutions at different concentrations was observed,indicating that water mobility was not affected by Gdm⁺or NH₄⁺.It reveals that Gdm⁺and NH₄⁺do not denature and stabilize protein structures through altering the water structure.The analysis on the change of T₂ of Gdm⁺or NH₄⁺in presence of different amino acids reveals that Gdm⁺binds to thiol group or hydroxyl group in the amino acid side chains,while NH₄⁺prefers to bind to the?-carboxyl group but slightly interacts with thiol group or hydroxyl group.Thus,the direct interaction between Gdm⁺and the sulfur atom or disulfide bond in proteins promotes the binding of Gdm⁺to proteins,leading to protein denaturation.(3)Unfolding of peptides LK₇ ?at the air-water interface was characterized by achiral and chiral sum frequency generation(SFG)spectroscopy.For urea concentration lower than 4.0 M,urea molecules accumulated at the air-water interface.As a result,the hydrogen bonding networks of water at the interface was disrupted.Urea disrupted the chiral hydration structure around LK₇?and induced?-sheet unfolding simultaneously.The collapse of the chiral hydration structure is assumed to promote the unfolding of?-sheet.For urea concentration higher than 4.0 M,urea molecules assembled at the air-water interface in a manner like solid urea crystal.?-sheet of LK₇?showed no obvious change with the increase of urea concentration,indicating that the urea-induced LK₇?unfolding at the air-water interface is incomplete denaturation.Accordingly,the air-water interface can help to stabilize the?-sheet of LK₇?.(4)The influence of chiral environment on the interfacial behavior of p H low insertion peptide(pHLIP)at the water-phospholipid bilayer interface was characterized by circular dichroism(CD)spectroscopy,fluorescence spectroscopy,fluorescence resonance energy transfer(FRET)and fluorescence quenching experiment.1,2-Dipalmitoyl phosphatidylcholine(L-DPPC)and its enantiomer(D-DPPC)small unilamellar vesicles(SUVs)with a diameter of100 nm were prepared by extrusion.Tryptophan fluorescence spectra showed that pHLIP adsorbed to the surface of two DPPC isomer SUVs.CD spectra showed that pHLIP folded into an?-helix structure at p H 4 at the water-phospholipid interface.Combining the results of FRET and fluorescence quenching experiments,it was found that pHLIP did not pass through the DPPC bilayer after its insertion.During the insertion of pHLIP,the chiral environment of the DPPC bilayer show slight impacts on the interfacial behaviors of pHLIP.