Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies

Abstract

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Figure 1

In vitro chemical ligation of folded recombinant proteins is illustrated by the preparation of Abl-SH(32). The Abl-SH3 domain is generated as an ethyl α-thioester derivative from the corresponding intein fusion protein, and the Abl-SH2 domain is generated with a cysteine at the N terminus via a factor Xa proteolysis strategy. Note that the linkage between the SH3 domain and the fused intein protein-splicing domain is naturally in equilibrium between an amide and a thioester (–17). Exposure of this fusion protein to ethanethiol at pH 6.0 results in the formation of an ethyl α-thioester derivative of the SH3 domain. Combining these SH3 and SH2 protein derivatives under conditions that maintain them as folded results in a chemoselective ligation reaction and the generation of a normal peptide bond at the ligation junction (26). The sequence of the final ligation product is m{65}LFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWAEAQTKNGQGWVPSNYITPVGCLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKR{220}gihrd. Lowercase letters indicate nongene residues from the expression systems used. This construct uses a C¹⁰¹ → S mutation internal to the SH3, which had previously been inserted to improve stability for NMR experiments. This is also in the “wild-type” sequence. Note that native chemical ligation reactions can be performed in the presence of multiple internal cysteine residues in either of the reacting segments (27); only the N-terminal cysteine participates in the ligation reaction.

Figure 2

Chemical ligation of Abl-[G¹²⁰]SH3 to Abl-[C¹²¹][U-¹⁵N]SH2. (A) Analytical reverse-phase HPLC profile of the crude ligation mixture after a 90-h reaction. A linear gradient of 32–46% of solvent B over 30 min was used. ESMS was used to identify the various components in the mixture, which are labeled accordingly. Note that the Abl-SH3 domain is converted to the more reactive benzyl- and phenyl-α-thioester derivatives in situ. (B) Electrospray mass spectrum (mass reconstruction) of the purified product, Abl-[G¹²⁰C¹²¹][SH2-¹⁵N]SH(32); expected mass (average isotope composition) = 18,240.2 Da.

Figure 3

¹H{¹⁵N} NMR spectra at 500 MHz of Abl-[G¹²⁰C¹²¹][SH2-¹⁵N]SH(32) (A) and wild-type Abl-SH(32) (B) with uniform ¹⁵N labeling. The peaks in A are the SH2-associated subset of those in B. (C–E) The peaks showing detectable chemical shift changes away from their position in the wild type are illustrated. (C) S¹²¹ in the wild type is mutated to C¹²¹ in the segment-labeled material. (C–E) The wild type subspectrum is shown in solid lines, and the segment-labeled protein is shown in dashed lines. Residue G¹³⁰ shows a small ¹H chemical shift (D), as does A¹⁹⁶ (E). Both of these residues are spatially close to the junction between SH3 and SH2 and presumably are slightly structurally perturbed.