Epitope scanning

Szabo, G., Pine, P., Weaver, J., Kasari, M. and Aszalos, A. (1992). Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system. Biophys. J. 61, 661-70. 

Fig. 5 Effect of varying relaxation delays between on- and off-resonance experiments in STD NMR experiments, a Experimental setnp for interleaved measnrements in STD NMR spectroscopy, n represents the nnmber of scans. The inter-scan delay Adi is varied while keeping on- and off-resonance freqnencies constant at -4 and -t300 ppm, respectively, b The resulting STD effects for the 0-methyl group of a-L-Fuc-O-methyl in the presence of RHDV VLPs. The equation that was used for non-linear least squares data fitting is based on the saturation recovery experiment [98], With Ti(iig) = 0.91 s as measured independently (unpublished results) and a Monte Carlo error estimation yields Ti(virus) = 10.06 0.41 s. This value does not directly correspond to a Tl relaxation time of the virus protons, because other factors also influence the observed relaxation [99]. According to these findings a relaxation delay Adi = 25 s was employed in all STD experiments. This results in a recovery of 92% of the virus resonance, and thereby reduces errors in epitope mapping that are introduced otherwise by non-homogeneous recovery of the binding site.

Wang, L F, Hertzog, P J, Galanis, M, Overall, M L., Waine, G J, and Linnane, A. W. (1994) Structure-function analysis of human IFN-alpha—Mapping of a conformational epitope by homologue scanning J Immunol 152, 705-715. 

Wang, B. L., Springer, G. F., and Harwick, L. C. 1998. T (Thomsen-Friedenreich) and Tn epitope location and their spatial relations to adhesion plaques on human breast carcinoma cells Immunogold silver staining studies at scanning electron microscopic level. Submicrosc. Cytol. Pathol. 30 503-509. 

Peptide Scanning Library (Epitope Mapping, Peptide Scan)... 

Peptide scanning is a very useful tool for screening a known protein sequence for active regions (e.g., epitopes). It was first described by Geysen and coworkers for peptides bound to polypropylene rods (28). The peptides are generated by shifting a frame of a distinct peptide length of a protein sequence of interest... 

A special application of the peptide scan is the hybritope scan (hybrid-epitope) (44,45). In order to increase the affinity by extention of the peptide sequence in a hybritope scan, several mixtures of amino acids at positions flanking the related sequence of the protein are introduced. Another strategy to optimize a peptide scan is the so-called duotope scan (duo-epitope) (46). For a duotope scan one generates the peptide scan twice and combines the sequence of both arrays linked via a spacer, such as two -alanine molecules. In this way it is possible to avoid possible steric hindrance and to increase activity by extention of the active sequence. The pattern of such a duotope scan resembles that of a combinatorial library with the exception that each field represents a combination of two sequences rather than two amino acids. 

Gunningham, B. C., and Wells, J. A. (1989). High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244, 1081-1085. 

The following studies were important because they represented another example of interpretation of cryo-EM results, using atomic models for the associated species. In addition, they confirmed and extended the results of natural escape mutation and peptide-scanning techniques used to identify the epitope region. 

Pal G, Fong SY, Kossiakoff AA, Sidhu SS. Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis. Protein Sci. 2005 14 2405-2413. Conlon JM. Evolution of the insulin molecule insights into structure-activity and phylogenetic relationships. Peptides 2001 22 1183-1193. 

Other, perhaps alternative, approaches that could be used for epitope mapping include homolog-scanning mutagenesis (5), which could also be a means of complementing the secondary structure predictions. This involves the synthesis of an array of small peptides that completely span the protein... 

Cunningham, B C.,Jhurani,P, Ng,P., and Wells, J A (1989) Receptor and anubody epitopes m human growth hormone idenufied by homolog-scanning muu enesis. Science 1330-1336. 

G.A. Weiss, G.K. Watanabe, A. Zhong, A. Goddard and S.S. Sidhu, (2000). Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc. Natl. Acad. 

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