Probing The Effect Of Metal Cations On The Abnormal Aggregation Of Amyloid-? Protein By AFM

Protein-protein interaction plays an essential role in the regulation of many biological functional processes.A number of human diseases are related to abnormal aggregating events of proteins and thus probing these interactions has become increasingly essential in the development of novel drugs.In recent years,Understanding of intermolecular interactions on a single molecule level by atomic force spectroscopy?AFM?is fundamentally important in the biomedical scienceand molecular biology.Some research suggested that the presence of metal cations is an important regulator in the abnormal aggregation processes of the amyloid ?-protein?A??,which have been proved to be an important pathological phenomenon in the brains of Alzheimer's disease?AD?patients.The previous literature focused on the studies by fluorescence spectroscopy and nuclear magnetic resonance.However,knowledge on the principle of interactions between A? and metal cations on a single molecule level is still poor understood.In this article,the role of metal ions on A? aggregation is studied by atomic force microscopy and discussed form the perspective of single molecular force with adhesion force and friction force measured.The following aspects of work were carried out mainly:?1?First of all,the A?42 was fabricated on thiol-modified gold substrates and gold-coated probe tip using self-assembled monolayer method?SAM?.The 16-Mercaptohexadecanoic acid?MHA?film was prepared by SAM method,the MHA then activated by 1-ethyl-3-?dimethylaminopropyl?carbodiimide hydrochloride?EDC?and N-Hydroxysulfosuccinimide?NHS?.The A?42 molecules were covalently bonded on the activated MHA film and a well-ordered protein monolayer was fabricated.Next,the obtained A?42 monolayer was characterized by tapping-mode atomic force microscopy?TM-AFM?,contact angle apparatus?CA?,X-ray photoelectron spectroscopy?XPS?and grazing incidence X-ray diffraction method?GIXRD?,respectively.In short,the results are as follows:1)The 3D topography of the bare gold and A?42 monolayer with different surface roughness and height recorded by AFM showed dissimilar nanostructures,indicating the A?42 molecules were successfully immobilized on the thio-modified gold surface.2)The contact angle results showed that the MHA film and the A?42 monolayer were all hydrophilic.In addition,the contact angles of mixed monolayers which formed by a series of molar ratio of MHA to dodecanethiol were also connected to the surface,the results show the contact angle was linear with the molar ration of dodecanethiol,but reversely linear with the molar ration of MHA.3)Moreover,the spectra of binding energy measured from these different surfaces could be well fitted with either Au4 f,S2p,or N1 s,respectively.4)The GIXRD 2? degrees of the MHA film and the protein monolayer ranged from 0° to 15°,significantly smaller than that of the bare gold surface,but the MHA film and the protein monolayer displayed very different profiles and distributions of their diffraction peaks.?2?The A?42 aggregation behavior was investigated by AFM in the liquid phase environment.The characteristics of A?42 monolayer in various conditions within 24 h,48 and 72 h were observed.The surface of A?42 monolayer in absence and presence of metal cations showed different nanostructure.The images of A?42 monolayer in the presence of metal cations showed accumulation characteristics with different nano structure and surface roughness.In the blank solution,no large gathering particles on the A?42 monolayer were observed with the increase of time;however,large gathering particles on the monolayer were generated in the presence of metal cations.The imaging results showed thatmetal cations accelerate the dimerization and oligomerization of A?42 molecules.?3?The adhesion forces in the longitudinal direction between A?42-A?42 in PBS and different kinds of metal ionic solutions were investigated by contact mode of AFM.The Possion statistical method was employed to determine the unbinding force of the single pair of A?42-A?42.The force results showed that the specific adhesion force Fi between a single A?-A? pair in control experiment was calculated as 42±3p N.However,Fi between a single A?-A? pair in the presence of Zn²⁺,Cu²⁺,Ca²⁺ and Al3+ increased dramatically to 89±3pN,84±6pN,73±5pN,95±5pN successively,which indicated that unbinding between A?42 molecules is accelerated in the presence of metal cations.It can be surmised that the increased adhesion force between A?42 molecules may be the important factor to cause dimerization and oligomerization of A?42.?4?The concentration of metal cations on the adhesive behavior between A?42-A?42 pair was also investigated.Meanwhile,the effect of metal cations on A?42 aggregation was discussed under various pH conditions.The results showed that increased concentration of metal cations promoted the specific interaction between A?42-A?42 pair.The specific force?Fi?between A?42-A?42 pair was significantly increased with higher metal cation concentration in the range of 1?M to 10?M.However,there is no significant change in Fi when the concentration is over 10?M.Study on the effect of metal cations on A?42 aggregation under various p H conditions showed that the presence of metal cations increased the specific force between A?42-A?42 pair when the pH value is over 5.0 and thus promoted the aggregation of A?42.But there was no effect of metal cations on Fi when pH value is less than 5.0.The result of imaging study in zinc solution also showed no aggregation nanoparticles with pH value less than 5.0.It follows that the protonation of proteininhibit the effect of metal ions on A?42 aggregation.?5?With respect to the FFM study of A?42-A?42 interaction in PBS and different kinds of metal ionic,friction forces between A?42 modified substrate and A?42 monolayer were investigated by lateral force mode.And the results showed the friction force between A?42 monolayer in the presence of metal cations mainly distributed in the range of 150 to 400 pN,with 50 to 150 pN in control group.It indicates that metal cations have significant influence on the friction force between A?42 monolayer.FFM study on A?42 monolayer connected to mixed mercaptan film of MHA/NDM indicated that the change of surface friction coefficient showed a similar trend to surface roughness,which maybe a main factor in diversity of friction.What's more,applied normal forces were tested to be linear with the friction forces.Both the result of friction force suggested that a certain correlation is existed between friction force and A?42 aggregation.?6?Surface enhanced Raman scattering?SERS?was applied to characterize the Raman intensity of A?42 monolayer in the absence and presence of metal cations.As to aluminium ion,Raman intensity peak of Amide II at 1375cm-1 in A?42 molecule is reduced considerably?almost disappeared?and peak at 1669cm-1corresponding to ?-sheet also decline compared with the blank control group,indicating that quite a number of amide linkage interact with Al3+ and cause structural change.Besides,A?42 monolayer in Zn²⁺ and Cu²⁺ solution presented a similar conformational transition characterized by Raman intensity with almost equal I1375/I1669.In Ca²⁺ solution,Raman intensity of ?-sheet declined compared with control group,but showed a smaller conformational transition than Zn²⁺ and Cu²⁺ group.The results studied by SERS are identical with AFM resuts.In conclusion,mechanical behavior between A?42 molecules and the effect of metal cations on A?42 aggregation was evaluated from the view of adhesive force and friction force with the help of SERS tracking technology.Atomic force microscopy extends the validation method of metal ions induction hypothesis on A?42 abnormal aggregation and it may provide helpful guide to developing new pharmaceutical treatment of disease.