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1.
Figure 2

Figure 2. From: Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing.

Semi-synthesis of Mxe GyrA intein constructs 1-12. (a) Schematic of the semi-synthetic route. (b) Summary of constructs used in the study, where R1 is the sequence N-terminal to the intein, X is the first intein residue, Z is the first C-extein residue and R2 is the sequence at the branch site. The chemical structures of the unnatural amino acids incorporated into the constructs are shown at bottom.

Silvia Frutos, et al. Nat Chem Biol. ;6(7):527-533.
2.
Figure 4

Figure 4. From: Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing.

Kinetics of the O-N acyl migration reaction. (a) Scheme showing the photoprotected O-acyl peptide (9) and its reaction. (b) Following irradiation peptide 9 was incubated in splicing buffer at pH 6.5. The conversion of unprotected ester peptide into amide product was followed by RP-HPLC. Shown are a series of RP-HPLC chromatograms of the reaction mixtures at different time-points in the absence of intein (c) Plot of the fraction amide product formed at different timepoints in the presence or absence of added intein.

Silvia Frutos, et al. Nat Chem Biol. ;6(7):527-533.
3.
Figure 1

Figure 1. From: Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing.

The mechanism of protein splicing. (a) Schematic illustrating conserved regions within the intein family. Conserved sequences (A, B, F and G) are indicated by filled boxes. Residues involved in the splicing reaction are shown below the bar. (b) Schematic of the mechanism of protein splicing. (c) Upper panel; ribbon representation of the Mxe GyrA intein structure (pdb code 1AM2) showing the location of Ala185 ligation site used for EPL. Lower panel; close up of the Mxe GyrA active site. His187 and 197 are shown in red, Asn198 in blue, Ser 1 in orange, Phe194 in purple and Val195 in yellow.

Silvia Frutos, et al. Nat Chem Biol. ;6(7):527-533.
4.
Figure 5

Figure 5. From: Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing.

Solution NMR analysis of isotopically labeled constructs. (a) Schematic representation of linear (10) and branched (11) constructs with the scissile +1 peptide bond site-specifically labeled with 13C and 15N. The recombinant component of the constructs was also labeled with 15N. (b) 1D HNCO spectra of construct 10 (left) and construct 11 (right) at pH 7.5 and 4 °C. The signal corresponding to the scissile +1 peptide bond could only be observed for the linear construct 10. Spectra were recorded on a 900 MHz spectrometer. (c) 1D HNCO spectrum of construct 11 under denaturing conditions (10% ACN/D2O + 0.1 %TFA).

Silvia Frutos, et al. Nat Chem Biol. ;6(7):527-533.
5.
Figure 3

Figure 3. From: Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing.

Functional characterization of branched and linear Mxe GyrA intein constructs. (a) Solution 1H{15N} HSQC spectra of constructs 1 (red) and 3 (black) recorded on a 900 MHz spectrometer at 4 °C. (b) Kinetics of succinimide formation for branched construct 1 (left) and linear construct 3 (right) at 25 °C and pH 7.5. Shown are a series of RP-HPLC chromatograms of the reaction mixtures at different time-points. Identity of starting materials and product were determined by ms.

Silvia Frutos, et al. Nat Chem Biol. ;6(7):527-533.
6.
Figure 6

Figure 6. From: Branched Intermediate Formation Stimulates Peptide Bond Cleavage in Protein Splicing.

Effect of changing pH, temperature and magnetic field on the solution structure of the +1 amide bond in the branched intermediate. (a) Schematic representation of site-specifically labeled construct 12. The protein contains 13C and 15N isotopes at both the scissile +1 amide bond (labeled b) and the amide connecting Phe194 and Val195 (labeled a). (b) 1D HNCO spectra of construct 12 at different pH recorded on a 900 MHz spectrometer. In the series, the temperature was kept constant at 4 °C, but the pH switched between pH 7.5 (green), pH 4.5 (black) and then back to pH 7.5 (red). (c) 1D-1H{15N} HSQC spectra of 12 at different temperatures recorded on a 900 MHz spectrometer. In the series, the pH was kept constant at pH 7.5, but the temperature increased from 4 °C (green), to 15 °C (black) and then to 25 °C (red). Peak assignments were based on a 1H{15N} HSQC-TOCSY experiment. (d) 1H{15N} HSQC spectra of 12 at different field strengths. In the series, the temperature and the pH were kept constant at 4 °C and 7.5 respectively, but the spectrum was acquired on a 500 MHz (red), 700 MHz (black) or 900 MHz (green) spectrometer.

Silvia Frutos, et al. Nat Chem Biol. ;6(7):527-533.

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