Design, total chemical synthesis, and binding properties of a [Leu-91-N1-methyl-7-azaTrp]Ras-binding domain of c-Raf-1

Abstract

The Ras-binding domain (RBD) of c-Raf-1 has been synthesized chemically, taking advantage of the chemical ligation of two peptide fragments of the protein. This procedure allowed incorporation of an unnatural amino acid (N1-methyl-7-azatryptophan) at position 91 of RBD, producing a protein with fluorescent properties distinct from and distinguishable from those of proteins containing the natural fluorophore tryptophan. The resulting protein was shown to interact with Ras in a manner that was almost indistinguishable from that of unmodified RBD based on transient kinetic monitoring of the binding event. Modified RBD containing the L-isomer of the unnatural amino acid or its racemic D,L mixture appeared to interact identically with Ras. The approach demonstrates a general procedure for the introduction of unnatural amino acids that can be used for monitoring protein-protein interactions and for the introduction of an unnatural backbone structure at strategic positions.

Figure 1

Ribbon presentation of the Rap1A-RBD complex (6). Leucine 91 (L91) is shown as a stick model. Figure was drawn with molscript (39).

Figure 2

Experimental strategy for the total chemical synthesis of RBD proteins. Two peptide segments of the target polypeptide are synthesized by optimized solid-phase synthesis protocols (28) and coupled by native chemical ligation at the ligation site Cys-95–Cys-96 (16, 18). Modifications are in bold. COSR, C-terminal thioester. O, optical probe, in this case N¹-methyl-7-azatryptophan, where D,L-O is the racemate and L-O is the l-enantiomer. Numbering is taken from the sequence of c-Raf-1.

Figure 3

Characterization of purified [L91D,L-O]RBD/H, the artificial protein with N¹-methyl-7-azatryptophan as a racemate at position 91 and a C-terminal His tag. (a) Chromatogram after purification on a preparative C4 RP-HPLC column. The chromatogram was obtained on an analytical C4 RP-HPLC column with a gradient of 5–65% acetonitrile in H₂O, 0.1% trifluoroacetic acid over 30 min. (b) Electrospray ionization-mass spectrometry of the purified product with the 5H⁺–11H⁺ charged states of the polypeptide corresponding to a measured mass of 10,544.5 ± 1.1 Da (calculated mass with an average isotope composition: 10,544.1 Da).

Figure 4

(a) Far-UV CD spectra of chemically synthesized proteins (csRBD/H, cs[L91D,L-O]RBD/H) and RBD proteins expressed in E. coli (RBD/H, [L91W]RBD/H). Spectra were obtained at 20°C in 20 mM phosphate, pH 7.4 with a protein concentration of 95 μM. (b) Fluorescence spectra of cs[L91L-O]RBD/H and biosynthetic [L91W]RBD/H were measured at 25°C in 100 mM NaCl, 50 mM Tris⋅HCl, 5 mM MgCl₂, pH 7.4 with a protein concentration of 2 μM. The excitation wavelength was 290 nm.

Figure 5

Increasing fluorescence transient observed on rapid mixing of [L91D,L-O]RBD/H (0.5 μM) with H-Ras.mantGppNHp (2 μM) and transient observed for the rapid mixing of [L91W]RBD/H (0.5 μM) with H-Ras.mantGppNHp (2 μM) at 25°C under the same conditions. The excitation wavelength was 313 nm with detection through a 360-nm cutoff filter. The fitted curve of the increasing transient corresponds to a pseudo-first-order rate constant of 90 s⁻¹.