1. Volume 8, Issue 4, Pages 413-425 (April 1998)
Alanine Scanning Mutagenesis of an αβ T Cell Receptor: Mapping the Energy of Antigen Recognition 
Thomas C Manning, Carol J Schlueter, Thomas C Brodnicki, Evan A Parke, Jeffrey A Speir, K.Christopher Garcia, Luc Teyton, Ian A Wilson, David M Kranz 
Immunity 
Volume 8, Issue 4, Pages (April 1998)
DOI: /S (00)

2. Figure 1 Antibody Reactivity of Representative scTCR Alanine Mutants
Wild-type or mutant scTCR preparations at various concentrations were used to inhibit the binding of biotinylated wild-type scTCR to the Vβ8-specific mAbs KJ16 (A), F23.1 (B), or F23.2 (C) in a competition ELISA. Bound biotinylated scTCR was detected by streptavidin-HRP. Reactivity of scTCR mutants to the clonotypic mAb 1B2 (D) was assessed in a capture ELISA format involving immobilized 1B2 and employing KJ16 as the detecting agent. The degree of reactivity of each mutant toward a specific antibody was determined as the IC50mut/IC50wt using linear regression analysis. In 1B2 ELISAs, the concentration of TCR required for 50% maximal signal was used analogously to the IC50.
Immunity 1998 8, DOI: ( /S (00) )

3. Figure 2 Summary of scTCR Binding to F23.2, 1B2, and QL9/Ld
Reactivities were calculated based on the relative IC50 of each mutant as compared to the wild type using linear regression analysis. Mutants that have decreased binding affinities compared to wild-type TCR are indicated by bars extending above the zero line, while mutants that have increased binding affinities are indicated by bars extending below the line. QL9/Ld assays were performed in triplicate and ELISAs in duplicate. Error bars represent the standard deviation obtained from two or more separate experiments. Mutants for which QL9/Ld binding was not detectable were considered as having at least 15-fold reduced binding (i.e., 49αTyr, 50αTyr, 31βAsn, 48βTyr, and 50βTyr).
Immunity 1998 8, DOI: ( /S (00) )

4. Figure 3 Structural Analysis of the 2C TCR Antigen Binding Site
Highlighting CDR loops (A), F23.2 epitope (B), 1B2 epitope (C), and QL9/Ld epitope (D). Colors shown reflect the reactivities presented in Figure 2, with red indicating residues that have the greatest influence on antigen binding, pink those with a moderate influence, yellow those with a negligible influence, and green indicating a mutant with significantly improved binding. The 2C TCR surface depicted is from the 2C/dEV8/Kb complex.
Immunity 1998 8, DOI: ( /S (00) )

5. Figure 4
Inhibition of 125I Anti-Ld Fab Binding by Wild-Type and Representative Alanine Mutant scTCRs
T2-Ld cells that had been loaded with the peptide QL9 were incubated for 1 hr at 4°C with 125I Fab fragments and various concentrations of inhibitor scTCR. Titrations were performed in triplicate, and bound counts per minute were determined after spinning cells through oil to separate free 125I Fab fragments. QL9-Ld reactivity for each mutant was determined as the IC50mut/IC50wt. These values were normalized for the extent of proper refolding, using Vβ8 reactivity for each mutant. Representative results of several different experiments are illustrated.
Immunity 1998 8, DOI: ( /S (00) )

6. Figure 5
Analysis of the Binding Energetics of TCR Residues in the 2C/QL9/Ld Modeled Complex
Residues evaluated for binding to QL9/Ld are colored according to the magnitude of their effect on ligand binding: red, greater than 8-fold reduced; pink, 2- to 8-fold reduced; yellow, 0.7- to 2-fold reduced; and green, less than 0.7-fold reduced (i.e., higher affinity). (A) Side view with the Ld α2 helix in the foreground. (B) Top view with the complex rotated approximately 90° toward the viewer, about an axis passing through peptide QL9. Energy calculations provide an estimate that approximately 37% of the energy is attributable to interactions with the peptide and approximately 63% to interactions with the antigenic MHC helices.
Immunity 1998 8, DOI: ( /S (00) )