Preparation, characterization and application of interleukin‐1β mutant proteins with surface‐accessible cysteine residues

Abstract

Two mutants of interleukin-la (K27C and K138C) were produced using site-specific mutagenesis in which lysine residues at positions 27 and 138 of the mature protein sequence were substituted by cysteine residues. The conformations of the mutant proteins were studied by 'H-NMR spectroscopy and shown to be similar to the wild-type protein. The receptor-binding affinity and biological activity of K27C and K138C were also similar to wild-type protein. The substituted cysteines in both mutant proteins were shown to be solvent-accessible as judged by their reactivity towards sulfhydryl reagents. As the wild-type protein contains two cysteines, which are both solvent-inaccessible in the native state, the mutants offer the opportunity to introduce probes in a sequencespecific manner via reaction with sulfhydryl groups. Examples of this are described in which the K138C was disulfide-linked to phycobiliproteins. The highly fluorescent conjugates had similar receptor-binding affinities to that of the wild-type unconjugated protein and were found suitable for flow-cytometric analysis.Interleukin-I (IL-1) refers to at least two monocyte-derived proteins (IL-la and p) which display a wide range of biological activities, including fever induction and augmentation of lymphocyte proliferation (reviews [I, 21). IL-la and p are both produced as 31-kDa precursors which are processed to give distinct bioactive 17-kDa proteins [3]. Although IL-la and have a relatively low sequence similarity ( x 23%) they bind to the same cell receptor [4, 51 and appear to effect a similar range of biological activities [l, 21.The availability of pure and well characterized recombinant-produced IL-la and p [6 -81 has enabled structural studies to be carried out using 'H-NMR spectroscopy [9,10] and X-ray crystallography [ll]. The latter work has provided a 0.3-nm model of IL-111 and, in the near future, a refined structure will probably be described. Even with the structural information available, it is not possible to identify residues or regions of the IL-1 molecule important for receptor binding.Most of the information in this area has come so far from site- Ahhreviation.r. TL-I , interleukin-1 ; K27C and K138C, interleukin1j3 mutant proteins with cysteine substituted for lysine at positions 27 and 138, respectively; IL-l/LAF assay, lymphocyte-activating-factor assay; Nbs,, 5,5'-dithiobis(2-nitrobenzoic acid); HOHAHA spectroscopy, homonuclear two-dimensional Hartmann-Hahn spectroscopy. acid substitutions, extensions and deletions to the N-terminal region of IL-1p [13 -151.

Crystallographic refinement of interleukin 1 beta at 2.0 A resolution.

“…Site-directed mutagenesis of IL-1 has also been used to probe the molecule for residues that may be important for activity (20)(21)(22)(23)(24)(25)(26). Many studies were also able to show, either by proton magnetic resonance (20)(21)(22)26) or by circular dichroism (23), that the overall structure of the protein was largely unaffected by the mutations.…”

“…Many studies were also able to show, either by proton magnetic resonance (20)(21)(22)26) or by circular dichroism (23), that the overall structure of the protein was largely unaffected by the mutations. (28).…”

“…The side chains of both of these residues are fairly exposed to solvent. A very interesting finding is that the 240-kDa fluorescent protein complex B-phycoerythrin can be coupled to the Lys -* Cys-254 mutant without significant loss of receptor binding, although biological activity is reduced 50-fold relative to wild-type IL-1,8 (26). Lys-254 is quite distant from both Arg-120 (30 A) and His-156 (20 A).…”

“…A number of peptides (10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and mutants (20)(21)(22)(23)(24)(25)(26) of IL-1 have been made in an attempt to probe receptor interactions and to devise a blocking agent for IL-1-induced inflammation. The full three-dimensional structure of mature IL-1 would be of use in this work.…”

Protein-based tumor molecular imaging probes

“…Cysteine harbors the specific thiol side chain among the normal amino acids, which can react with various thiol-specific coupling reagents without affecting other amino acid species, with the premise, of course, that no other cysteine is present on protein surface. A surface-accessible cysteine can be introduced into target protein by site-directed mutagenesis (Wingfield et al 1989). For instance, scFvs have been cysteine modified with PEG to adjust the circulation half-life (Yang et al 2003).…”