| Proteins play essential role in biological process. Many biological functions are regulated or manipulated by protein-protein interactions. The study of interactions between antigen and antibody is one of the most improtant research fields of protein-protein interactions. In this work, two protein pairs, namely the insulin (INS)/insulin degrading enzyme (IDE) and the human IgG (antigen)/rat anti-human IgG (antibody) were selected to serve as model systems to investigate the protein-protein interactions. The research contents are as follows:(1) The interactions between INS (PDB access number: 2jv1) and IDE (PDB access number: 2jg4) were performed by a molecular simulation methods which is entitled as "Two-dimensional Graphics Lab for Biosystem Interactions". The results showed desolvation effect and Van der Waals interaction exsit during the binding process. As for the desolvation effect between INS and IDE, the desolvation free energy contributed by chain A and chain B of the complex are -4.288 kcal/mol and -5.495 kcal/mol, respectively. With respect to the Van der Waals interaction, chain A and chain B of the complex contribute -0.199 kcal/mol and -0.249 kcal/mol to it, respectively. The summarized residue-pair diagram shows there was steric clash between the Thr55 in chain A and the Thr30, and the distance between centroids is 1.71 nm. There was ion-pair interaction between the His53 in chain A and the Glu4 in chain B, and the distance between centroids is 5.35 nm.The INS interface consists of hydrophobic amino acids, thus leading to the formation of a strong hydrophobic environment in the solution of its instability, and thus as an effective chemical force driving the occurrence of combination of INS and IDE at nanomolar level. Intensive intermolecular contact had taken place in the interface of IDE and INS, resulting in an apparant matrix mosaic site and formed a pair of electrostatic interaction of the salt bridge. At the same time, under the collaborative drving of the shape complementarity of van der Waals and hydrophobic forces, IDE and INS formed the transient and stable complex structure, which mediates the downstream biological effects.(2) The 16-Mercaptohexadecanoic acid (MHA) film was prepared by self assembled method (SAM), the MHA then activated by 1-Ethyl-3- (Dimethy laminopropyl) Carbodiimide Hydrochloride (EDC) and N- Hydroxysulfosuccinimide (NHS). The antibody molecules were covalently linked on the activated MHA film and a well ordered antibody monolayer was fabricated.The obtained antibody monolayer was characterized by tapping-mode atomic force microscopy (TM-AFM), grazing incidence X-ray diffraction method (GIXRD), X-ray photoelectron spectroscopy (XPS) and contact angle genometry (CA) measurements, respectively. Both the 2D and 3D topographies of the bare gold, MHA film and the antibody monolayer were recorded by AFM, and they showed dissimilar nanostructures. The GIXRD 2θdegrees 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. Moreover, the spectra of binding energy measured from these different surfaces could be well fitted with either Au4f, S2p, or N1s, respectively. The Au4f spectra showed chemical shifts after exposure to MHA solution. With respect to S2p spectra, no detectable peaks above 164 eV were found. This means that no unbound thiol molecules presented on the MHA film. The contact angle of the MHA film and the protein monolayer were 18°and 12°, respectively, all being hydrophilic.In addition, the contact angles of mixed monolayers which formed by a series of molar ratio of MHA to dodecanethiol were also condected, the results showed the contact angle was linear with the molar ration of dodecanethiol, but reversely linear with the molar ration of MHA.(3) The interactions between antigen and antibody were imaged by TM-AFM and fiction force microscopy (FFM). As for the TM-AFM imaging, both the topographies and phase image of the bare gold, antibody monolayer and the antigen/antibody complexes were recorded by TM-AFM, respectively. The images of different surfaces showed different structure, indicating the antibody molecules were successfully immobilized on the thiol-modified gold surface and the complexes were formed due to the specific interaction between antigen and antibody.With respect to the FFM imaging, the topographies of the bare gold, the antibody monolayer, the antigen/antibody complexes, the blocking experiment and the reverse experiment were all recorded. Different surfaces showed different nanostructure, and quite in agreeable with prediction. This result suggested there are specific interaction between antigen and antibody. The pleatu-linked structure of blocking experiment was caused by the broadening effect of AFM tip. Notably, comparing to TM-AFM, the FFM measurements have better reproducibility. (4) The adhesive forces and friction forces between antigen and antibody were investigated. The adhesive force of antigen modified tip/antibody monolayer, bare tip/antibody monolayer, blocking experiment and reverse experiment were 0.6-1.0 nN, 0-0.2 nN, 0-0.2 nN and 1.0-1.2 nN, respectively, suggesting the specific interaction existed between antigen and antibody. The Possion statistical method was employed to determine the unbinding force of the single pair of antigen/antibody, and the unbinding forces and the nonspecific interaction forces was calculated to be 144±11 pN and 69 pN, respectively. Moreover, the possible artifacts during adhesive forces measurements were discussed, and the emprical solution were also provided.The friction forces between antigen and antibody were studied by FFM, the results showed the friction forces were linear with the applied normal forces. The friction forces of antigen modified tip/antibody monolayer, bare tip/antibody monolayer, blocking experiment and reverse experiment were 200-250 pN, 0-50 pN, 50-150 pN and 250-300 pN, respectively. The friction forces were one magnitude smaller than the adhesive forces. The results indicated specific interation forces between antigen and antibody. However, the detected large friction forces of bare gold/antibody monolayer may contribute to the material nature of AFM tip.The abovementioned results demostrated that 2D-GraLab is a powerful tool in computational simulating the protein-protein interactions, and it may provided helpful guide to the experimental studies. The SAM method is well suitable to immobilize the protein molecules. Besides, it is reliable and easy to do. Both the TM-AFM and FFM imaging are effective techniques to study the interaction between antigen and antibody. Both the adhesive forces and friction forces between antigen and antibody were revelaed by the AFM. Taking together, studies invovled the protein-protein interactions at the molecular level from the imaging and force points of view provide fundermental knowledge of biosensors, biomaterials and other biomedical research fields.
|
PDF Full Text Request