Results: 4

1.
Figure 1

Figure 1. From: Teaching an old scaffold new tricks: Monobodies constructed using alternative surfaces of the FN3 scaffold.

Monobody library design. (A) A comparison of the VHH scaffold (left) and the FN3 scaffold (right). The two β-sheet regions are colored in cyan and blue, respectively. The CDR regions of the VHH and the corresponding loops in FN3 are colored and labeled. The β-strands of FN3 are labeled with A–G. (B) The structure of a monobody bound to its target, maltose-binding protein.12 The monobody is depicted in the same manner as in A. Only a portion of maltose-binding protein is shown as a surface model. (C) The structure of a monobody bound to the Abl SH2 domain depicted as in B.15 (D) The locations of diversified residues in the “loop only” library shown as spheres on the FN3 structure. (E) The locations of diversified residues in the “side and loop” library.

Akiko Koide, et al. J Mol Biol. ;415(2):393-405.
2.
Figure 4

Figure 4. From: Teaching an old scaffold new tricks: Monobodies constructed using alternative surfaces of the FN3 scaffold.

The crystal structures of monobodies originating from the two libraries. The structures are shown with the monobodies in similar orientations. (A) The structure of the SH13 monobody bound to the Abl SH2 domain depicted as in Fig. 1C. (B) NMR-based epitope mapping of the SH13/Abl SH2 complex. The red, yellow and gray spheres show residues of Abl SH2 whose amide resonances were strongly affected (shift of >1.5 peak width), weakly affected (shift of 0.5–1.5 peak width) and minimally affected (shift of <0.5 peak width) by monobody binding, respectively. (C) The crystal structures of the ySMB-1/ySUMO complex (left) and ySMB-9/hSUMO1 complex (right). (D) The two SUMO-binding monobodies bound to equivalent epitopes on the targets using distinct modes. The top panel shows a comparison of the two crystal structures shown in C with ySUMO and hSUMO1 superimposed. The bottom panel shows ySUMO and hSUMO1 in equivalent orientations with the epitopes for the indicated monobodies highlighted in green and blue, respectively. Epitope residues are defined as those containing at least one non-hydrogen atom within 5 A of a non-hydrogen atom of the bound monobody in the crystal structures. (E) Alignment of portions of the ySUMO (top) and hSUMO1 (bottom) sequences indicating epitopes for ySMB-1 and ySMB-9, respectively. Residues in the ySMB-1 epitope are shaded in blue, and residues in the ySMB-9 epitope in green. Residue numbering is according to the ySUMO sequence.

Akiko Koide, et al. J Mol Biol. ;415(2):393-405.
3.
Figure 3

Figure 3. From: Teaching an old scaffold new tricks: Monobodies constructed using alternative surfaces of the FN3 scaffold.

Oligomerization state and stability of monobodies. (A) Size-exclusion chromatograms of monobodies. The chromatographs are shown with vertical offsets for clarity. The labels show the identities of analyzed samples. The void volume (V0) and elution positions for bovine serum albumin (BSA; 67 kDa) and ribonuclease A (RNaseA; 13.7 kDa) are indicated with the arrows. The monobodies have an average molecular weight of ~14 kDa. The GS2 monobody is exhibited a monodispsersed peak but appeared to interact with the chromatography media, resulting in delayed elution. (B) Fluorescence emission intensity of monobodies plotted as a function of guanidine thiocyanate concentration. The emission wavelengths that gave good transitions were different for different monobodies, and they are indicated on the vertical axis. The curves are the best fit of the two-state transition model. The vertical scales of the data for AS15[H33R] and AS27[Y33R] have been adjusted so that their native and denatured baselines coincide with the counterparts of their respective parent proteins. The denaturant concentration at the mid point of the denaturation transition (C0.5) is shown for each protein. The errors are the standard deviations from curve fitting of the two-state model.

Akiko Koide, et al. J Mol Biol. ;415(2):393-405.
4.
Figure 2

Figure 2. From: Teaching an old scaffold new tricks: Monobodies constructed using alternative surfaces of the FN3 scaffold.

Monobody library designs and generated clones. Amino acid sequences of monobodies generated from the new “side and loop” library (A) and the “loop only” library (B). “X” denotes a mixture of 30% Tyr, 15% Ser, 10% Gly, 5% Phe, 5% Trp and 2.5% each of all the other amino acids except for Cys; “B”, a mixture of Gly, Ser and Tyr; “J”, a mixture of Ser and Tyr; “O”, a mixture of Asn, Asp, His, Ile, Leu, Phe, Tyr and Val; “U”, a mixture of His, Leu, Phe and Tyr; “Z”, a mixture of Ala, Glu, Lys and Thr. (C) Binding measurements by yeast surface display of representative monobodies. The mean fluorescence intensities of yeast cells displaying a monobody are plotted as a function of the concentration of the target as indicated in panel A. The errors indicated are the standard deviations from curve fitting of the 1:1 binding model. A portion of data for point mutants of As15 and As27 is shown for clarity. (D) SPR sensorgrams for target binding of representative monobodies. The thin lines show the best global fit of the 1:1 binding model. The insets show dose-dependence analysis of the sensorgrams (black) and the best fit of the 1:1 binding model (gray). The errors indicated are the standard deviation of the global fits from at least triplicate data sets for the kinetic experiments, or the standard deviation from global curve fitting of duplicate data sets for the equilibrium experiments.

Akiko Koide, et al. J Mol Biol. ;415(2):393-405.

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