SpringerImages - 2D 1H-13C HSQC-NOESY experiments using deuterated FAS-TE with 15N/13C-labeled p-OMePhe incorporated at selected positions. a. Structure of the orlistat-FAS-TE complex (2PX6.pdb) (Pemble et al. 2007). Missing loops are indicated with a dashed line and orlistat is shown in magenta lines. The sidechain positions of the different single mutants are shown in red. b. Tool compound used in binding studies. c. 2D HSQC-NOESY spectra (Fesik and Zuiderweg 1988) for the indicated mutants. The spectra for each mutant before (blue) and after compound addition (red) are overlaid. The only exception is Tyr-2343 which did not have an HSQC peak in the absence of compound. The intense aromatic peaks correspond to intramolecular peaks between the 13C methoxy group and the sidechain. Arrows mark intermolecular correlations from p-OMePhe to the tool compound. The additional cross peaks were also found in HSQC spectra (see Supplemental Fig. 1) and correspond to natural abundance 13C peaks from contaminants (glycerol, tris, TCEP) in the buffer. In the case of the Tyr-2347 mutant protein, the limited solubility of the tool compound resulted in the presence of some residual apo protein

Caption

Fig 2

2D ¹H-¹³C HSQC-NOESY experiments using deuterated FAS-TE with ¹⁵N/¹³C-labeled p-OMePhe incorporated at selected positions. a. Structure of the orlistat-FAS-TE complex (2PX6.pdb) (Pemble et al. 2007). Missing loops are indicated with a dashed line and orlistat is shown in magenta lines. The sidechain positions of the different single mutants are shown in red. b. Tool compound used in binding studies. c. 2D HSQC-NOESY spectra (Fesik and Zuiderweg 1988) for the indicated mutants. The spectra for each mutant before (blue) and after compound addition (red) are overlaid. The only exception is Tyr-2343 which did not have an HSQC peak in the absence of compound. The intense aromatic peaks correspond to intramolecular peaks between the ¹³C methoxy group and the sidechain. Arrows mark intermolecular correlations from p-OMePhe to the tool compound. The additional cross peaks were also found in HSQC spectra (see Supplemental Fig. 1) and correspond to natural abundance ¹³C peaks from contaminants (glycerol, tris, TCEP) in the buffer. In the case of the Tyr-2347 mutant protein, the limited solubility of the tool compound resulted in the presence of some residual apo protein

Extracts from the Article What's this?

To validate this approach, fully deuterated FAS-TE mutants were produced with a single ¹⁵N/¹³C-labeled p-OMePhe residue incorporated (Fig. 2).

2D ¹H-¹³C HSQC-NOESY spectra (Fesik and Zuiderweg 1988) were recorded for selected mutants before and after the addition of a tool compound (Fig. 2). .

Additional correlations (see arrows in Fig. 2c) for the Leu-2222, Tyr-2343, and Tyr-2347-p-OMePhe mutants appear upon compound binding and represent inter-molecular NOE peaks from the unnatural amino acid to the tool compound.

Viewing this image requires a subscription. If you are a subscriber, please log in.

Other Images from this Article

License

This image is copyrighted by Springer Science+Business Media B.V..

The image is being made available for non-commercial purposes for subscribers to SpringerImages. For more information on what you are allowed to do with this image, please see our copyright policy.

If you would like to obtain permissions for the re-use or re-print of this image, please click here.