Directed Evolution of the Suicide Protein O⁶-Alkylguanine-DNA Alkyltransferase for Increased Reactivity Results in an Alkylated Protein with Exceptional Stability

Abstract

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O 6 -alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure−function relationship of the DNA repair protein.T he specific labeling of proteins with synthetic probes is a powerful approach for studying protein function. One way to achieve such a specific labeling is based on so-called selflabeling protein tags.1 In this approach, the protein of interest is expressed as a fusion protein with a peptide or protein (i.e., tag) whose role is to specifically bind to a synthetic probe in vitro or in vivo. A well-established example of a self-labeling protein tag is SNAP-tag.2 SNAP-tag specifically reacts with substituted O 6 -benzylguanine derivatives and thereby permits the labeling of SNAP-tag fusion proteins with a wide variety of different synthetic probes. Recent applications include its use for the analysis of protein complexes, 3 super-resolution microscopy, 4 the identification of protein−protein interactions, 5 drug target identification, 6 and the determination of protein half-life in animals. 7 The appeal of self-labeling tags such as SNAP-tag is the ease with which fusion proteins can be labeled with synthetic probes even in living cells. A conceptual limitation of the approach is the fact that the tag can affect the properties of its fusion partner. It is therefore important that the properties of the tag be as thoroughly characterized as possible.SNAP-tag was generated in a stepwise manner from human O 6 -alkylguanine-DNA alkyltransferase (hAGT) by introduction of a total of 19 point mutations (Figure 1) and deletion of 25 C-terminal residues. Saturation mutagenesis of four active-site residues followed by phage display and selection for activity against BG derivatives resulted in GE AGT, a mutant with 20-fold increased activity toward such substrates ( Figure 1B). 8Subsequent saturation mutagenesis of four additional residues involved in substrate binding followed by phage selection resulted in AGT-54, a mutant with 1.5-fold higher activity than GE AGT. To further optimize the protein for applications in protein labeling, mutations were introduced to suppress DNA binding and reactivity toward nucleosides, to remove nonessential cysteines, and to truncate the last 25 residues. 9The resulting mutant M AGT displayed relatively low activ...

Protein sensing in living cells by molecular rotor-based fluorescence-switchable chemical probes

“…However, cellular alkylated SNAP-tag has been known to resist degradation compared to alkylated-MGMT. 41 Under the same imaging conditions, no obvious fluorescence reduction was observed for the nucleus localized SNAP-H2B proteins alkylated with BG-CCVJ (Fig. 5a, CHO SNAP-H2B).…”

In vitro affinity screening of protein and peptide binders by megavalent bead surface display

“…However, the K d for immobilized streptavidin is reduced compared with streptavidin in solution (Chivers et al , 2010), so BB primers were adopted in an effort to partially compensate for this loss of affinity (Dressman et al , 2003). In order to capture the POI that was in vitro expressed from the DNA template, the POI was fused with the SNAP-tag (AGT, O 6 -alkylguanine-DNA-alkyltransferase) (Keppler et al , 2003; Mollwitz et al , 2012) that reacts with the BG forming a covalent thioether bond (Fig. 3).…”

“…The plasmid pIVEX-SNAP-HA was derived from pIVEX-SNAP-GFP (Keppler et al , 2003; Mollwitz et al , 2012) by double digestion with NotI and BamHI and subsequent ligation with T4 DNA ligase (1 h, room temperature) to the overlapping oligonucleotides F-HA and R-HA coding for the HA-tag (Supplementary Table S3). F-HA and R-HA were mixed and incubated with a ramp from 85°C to room temperature to let them anneal, before ligation into the digested vector.…”

Environment‐sensitive Fluorescent Turn‐on Chemical Probe for the Specific Detection of O‐Methylguanine‐DNA Methyltransferase (MGMT) in Living Cells

“…21,26 It has been previously reported that alkylated MGMT is more sensitive to proteases such as trypsin. 27,28 The increased protease susceptibility can be interpreted as a decreased stability of the alkylated protein. Currently, the alkylated-MGMT degradation kinetics can be obtained by detecting alkylated MGMT protein fragments via SDS-PAGE gels.…”