Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library - PubMed (original) (raw)

. 2010 Dec 1;132(47):16812-7.

doi: 10.1021/ja105429n. Epub 2010 Nov 10.

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Identification of highly reactive sequences for PLP-mediated bioconjugation using a combinatorial peptide library

Leah S Witus et al. J Am Chem Soc. 2010.

Abstract

Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We have previously reported a pyridoxal 5'-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine-lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

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Figures

Figure 1

Figure 1

The general scheme of PLP-mediated bioconjugation. (a) In the first step, a protein is incubated with PLP (1) under mild, aqueous conditions. This oxidizes the N-terminus of the protein to a ketone or an aldehyde, providing a unique functional group for further modification. (b) In the second step the ketone is conjugated to an alkoxyamine-bearing reagent (2) through oxime formation. (c) The proposed mechanism begins with Schiff base formation between the N-terminal amine and the PLP aldehyde. Tautomerization, followed by hydrolysis, affords the keto-protein product. Based on this mechanism, the reaction rate is expected to depend on both the concentration of protein and PLP.

Figure 2

Figure 2

Library screening scheme. (a) The 8,000-member combinatorial peptide library of the form XXXWSNAG was subjected to PLP-mediated transamination and subsequent oxime formation with Disperse Red alkoxyamine 2a. Stringent reaction conditions were used during the PLP reaction such that only sequences with high reactivity transaminated to form a keto group. These active sequences were colorimetrically distinguished from the others during the next step, where the Disperse Red dye formed a covalent oxime linkage with the transaminated sequences. The library was then examined under a microscope, and the red beads were manually removed for sequencing. (b) Each bead contained a truncation ladder in addition to the full-length peptide that had participated in the reaction. Sequencing was performed by cleaving the peptide species from selected beads and identifying the ladder peptides using MALDI-TOF MS. The mass differences between adjacent capped species corresponded to the amino acid in that position. Capped species were easily identified by the unique isotope pattern of the bromine atoms. The mass of the uncapped peptide can be identified at 824 m/z, confirming the assignment. The stuctures of the bromobenzoic acid caps 3 and 4, and the Disperse Red alkoxyamine used in these experiments are shown in (c).

Figure 3

Figure 3

Visual evaluation of the library at different screening conditions, including sequences identified from a 100 μM PLP reaction. (a) When incubated with 10 mM PLP, the majority of the library members turned red, confirming that at standard reaction conditions many sequences exhibit at least some degree of conversion. (b) Using 1 mM PLP, fewer library members turned red, and most showed a lighter color. (c) Using only 100 μM PLP, most of the sequences showed no conversion (as evidenced by the colorless beads) but a small number of red beads were still identified. (d) The sequences corresponding to the red beads at 100 μM PLP revealed AXK and AKX as the predominant N-terminal motifs.

Figure 4

Figure 4

Analogs of the reactive alanine-lysine motif identified during library screening were synthesized to verify the reactivity of this sequence. AKT was compared to LKT to confirm the importance of alanine as the first residue and AET was used to probe the role of a positive charge in the second position. At 0.1 mM PLP, the concentration of library screening, AKT showed a significantly higher yield than the other sequences. AKT also resulted in notably improved yields at higher concentrations of PLP.

Figure 5

Figure 5

Positional scanning was performed to explore the scope of the AKX and AXK motifs. The total oxime yield after reaction with standard PLP conditions (10 mM, 18 h) was determined for 38 peptides with varied amino acids in the X positions (all natural amino acids except cysteine were included). Although high yields were seen in all cases, peptides with lysine in the second position consistently provided higher conversion.

Figure 6

Figure 6

The performance of wild-type and AKT-terminal sequences were compared for the transamination of two protein substrates. The positions of the new termini are shown for (a) an “Antifreeze” Protein (AFP) and (b) GFP. The Ala residue is in red and the Lys-Thr portion is in yellow. (c) The transamination of the AFP mutant showed that the AKT sequence outperformed the wild-type terminus (GNQ) at every time point analyzed. (d) The AKT-terminal AFP also outperformed the wild-type sequence at different concentrations of PLP. (e) The product distribution of the AKT-terminal AFP mutant shows that maximum oxime yield (blue plus green) and minimum PLP adduct (green plus pink) could be achieved by optimizing the reaction conditions. The dotted line marks the highest total oxime yield acheived, demonstrating that the strategies that decreased byproduct formation did not sacrifice overall yield. (f) Using the strategy of a high concentration of PLP for a short reaction time, conditions were found that gave a high oxime yield with minimal byproducts for AKT-terminal GFP in only 90 minutes. The wild type GFP mutant achieved no modification under the same conditions, highlighting the enhanced reactivity conferred by the AKT sequence.

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