Epitope binding

Hjorth S, Thirstrup K, Gandy D et al. Analysis of selective binding epitopes for... 

Figure 7.2 Schematic showing the relationship of the native antigen to the peptide mimic. The native antigen (a protein) is shown as a winding, twisted line, so as to represent a hypothetical three-dimensional structure. The peptide represents the antibody-binding epitope (shown in dotted lines) of the native antigen. The epitope can represent a linear sequence of the native protein. Alternatively, the epitope can be formed by amino acids that are not immediately adjacent to each other in the primary sequence but brought together by the three-dimensional folding of the protein. Adapted with permission from Sompuram et al.6...

STD experiments allow for a mapping of the binding epitope (group epitope mapping, GEM) [25]. The degree of saturation of ligand resonances depends on the distance of the protons involved. Protons in close proximity to the target molecule are saturated to a... 

Observation of chemical shift changes or selective line broadening of the ligand resonances upon addition of protein indicate which parts of the ligand are in contact with the protein. Residues that are unaffected in position and line width are probably not in contact with the protein, and are therefore candidates for linker attachment Alternatively, the binding epitopes can be mapped by STD-type experiments [19]. 

Our study underscores the significance of a quantitative interpretation of STD intensities using the CORCEMA-ST procedure for the characterization of binding epitopes at atomic resolution. In contrast to what was sometimes suggested [84], we show here that the STDs involving methyl resonances also give accurate information about epitopes in quantitative analyses. 

Fig.l Ribbon presentations of spatial structure of the TGF-P3 dimer. Secondary structure elements and chain termini for the one monomer are labelled on the top view. TGF-fi type 1 receptor binding epitope, designated as wrist , TGF-p type 11 receptor binding epitopes, named as knuckle/fingertips , and an additional thumb epitope are circumscribed (on one monomer) by dashed ovals. The S-S bonds are shown in black... 

Utilizing STD NMR experiments we have resolved the binding epitope for one of the WIN compounds, REPLA 394, at atomic resolution [53]. Competitive STD NMR titrations then yielded the dissociation constant. When bound to native human rhinovirus serotype 2 (HRV2), saturation is predominantly transferred to the isoxazole and the benzoic acid residues, whereas the aliphatic linker receives less saturation (Fig. 3). Results reported before [16] are in accordance with the data reported in our study. 

Fig. 6 Binding epitopes for the B-antigen trisaccharide in the presence of RHDV VLPs recorded with a Arfl = 20 xs and b Arfl = 100 s. A setting of Adi = 20 jis for the interpulse relaxation delay corresponds to a total relaxation delay t = 4.7 s since relaxation also occurs during the acquisition time. With Adi = 100 s an increase of STD effects for H2, HI, H4 and H6 " is observed. This is dne to rednced satimation dnring the off-resonance experiment resulting from a sufficient long relaxation delay...

Following these experimental guidelines it will be possible to analyze the binding epitopes of HBGA hgands of RHDV at atomic resolution. 

Liu et al. [140] have also used this interface for an electrochemical immunosensor for small molecules (Figure 1.26). In this sensor, one end of the molecular wire is attached to ferrocene dimethylamine with a covalent link formed between one of the amine group son the ferrocene and the carboxyl group on the wire. To the other amine is attached the antibody-binding epitope for the antibody, in this proof-of-concept study the epitope is biotin. Electron transfer can be readily achieved to the ferrocene molecule but upon antibody binding to this interface, the electrochemical signal is dramatically reduced. 

As a consequence, any nonspecific adsorption of protein to the interface will also cause an attenuation in electrochemistry. Hence, the antifouling layer is required to ensure the attenuation of the electrochemistry associated with the presence of antibodies is due to a specific interaction with the binding epitope on the molecular wire rather than any nonspecific effects. The ability of this layer to resist nonspecific adsorption of proteins, as well as preventing electroactive species reaching the electrode, was demonstrated by Liu and Gooding [131] previously and is demonstrated in the current paper via controls that show there is little attenuation in the ferrocene electrochemistry if a different antibody is used or if the biotin epitope is absent from the interface. 

Scheme 4.9 Kinetic and thennodynamic self-sorting based on guests with two binding epitopes.

Ishikawa, M., Ishida, M., Shimakura, K., Nagashima, Y., and Shiomi, K. (1998a). Tropomyosin, the major oyster Crassostrea gigas allergen and its IgE-binding epitopes. /. Food Sci. 63, 44-47. 

Cairo CW, Gestwicki JE, Kanai M, KiessUng LL. Control of multivalent interactions by binding epitope density. J Am Chem Soc 2002 124 1615-1619. 

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