Published on Web 11/10/2010 Identification of Highly Reactive Sequences For PLP-Mediated Bioconjugation Using a Combinatorial Peptide Library Leah S. Witus,† Troy Moore,† Benjamin W. Thuronyi,† Aaron P. Esser-Kahn,† Rebecca A. Scheck,† Anthony T. Iavarone,§ and Matthew B. Francis*,†,‡ Department of Chemistry, UniVersity of California, Berkeley, California 94720-1460, United States, Materials Sciences DiVision, Lawrence Berkeley National Laboratories, Berkeley, California 94720-1460, United States, and QB3/Chemistry Mass Spectrometry Facility, UniVersity of California, Berkeley, California 94720-1460, United States Received June 21, 2010; E-mail: [email protected] 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. Introduction the many instances of the same functional groups on protein Protein bioconjugates are increasingly being used as func- surfaces. Although this is acceptable for some applications, tional and structural components of new materials.1 In a typical improved site selectivity is required to access architecturally approach, a synthetic functional group of interest is attached to precise biomolecular materials. This is often achieved by a specific amino acid side chain using one of a relatively limited targeting cysteine residues, but there are cases in which it is set of chemical reactions that can modify unprotected biomol- inconvenient or impossible to introduce a single copy of this 2,3 residue without sacrificing protein function. Also, a growing ecules in aqueous solution. However, most strategies (such 4 as lysine targeting) lead to complex product mixtures due to number of applications (such as FRET-based sensors, pollutant responsive hydrogels,5 and therapeutic delivery systems6) require † Department of Chemistry, University of California, Berkeley. the modification of proteins in two distinct locations. While § QB3/Chemistry Mass Spectrometry Facility, University of California, cysteine modification may be used to install the first group, Berkeley. compatible modification methods that can be used to install the ‡ Lawrence Berkeley National Laboratories. second functional group are in short supply. (1) For lead examples, see: (a) Douglas, T.; Young, M. Nature 1998, 393, 152–155. (b) Abedin, M. J.; Liepold, L.; Suci, P.; Young, M.; Douglas, As one solution to this challenge, we have previously T. J. Am. Chem. Soc. 2009, 131, 4346–4354. (c) Lee, S.; Mao, C.; reported7-9 a protein bioconjugation reaction that achieves site- Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892–895. (d) Nam, Y. S.; Shin, T.; Park, H.; Magyar, A. P.; Choi, K.; Fantner, G.; Nelson, K. A.; Belcher, A. M. J. Am. Chem. Soc. 2010, 132, 1462–1463. (e) (4) Crochet, A. P.; Kabir, M.; Francis, M. B.; Paavola, C. D. Biosens. Wang, Q.; Lin, T.; Tang, L.; Johnson, J. E.; Finn, M. G. Angew. Chem., Bioelectron. 2010, 26,55-61. Int. Ed 2002, 41, 459–462. (f) Niemeyer, C. M. Angew. Chem., Int. (5) Esser-Kahn, A. P.; Iavarone, A. T.; Francis, M. B. J. Am. Chem. Soc. Ed. 2001, 40, 4128–4158. (g) McMillan, R. A.; Paavola, C. D.; 2008, 130, 15820–15822. Howard, J.; Chan, S. L.; Zaluzec, N. J.; Trent, J. D. Nat. Mater. 2002, (6) (a) Kovacs, E. W.; Hooker, J. M.; Romanini, D. W.; Holder, P. G.; 1, 247–252. (h) Wang, P.; Sergeeva, M. V.; Lim, L.; Dordick, J. S. Berry, K. E.; Francis, M. B. Bioconjugate Chem. 2007, 18, 1140– Nat. Biotechnol. 1997, 15, 789–793. (i) Heredia, K. L.; Maynard, H. D. 1147. (b) Tong, G. J.; Hsiao, S. C.; Carrico, Z. M.; Francis, M. B. Org. Biomol. Chem. 2007, 5, 45–53. J. Am. Chem. Soc. 2009, 131, 11174–11178. (2) Hermanson, G. T. Bioconjugate Techniques, 1st ed.; Academic Press: (7) Gilmore, J. M.; Scheck, R. A.; Esser-Kahn, A. P.; Joshi, N. S.; Francis, San Diego, CA, 1996. M. B. Angew. Chem., Int. Ed. 2006, 45, 5307–5311. (3) Tilley, S. D.; Joshi, N. S.; Francis, M. B. The Chemistry and Chemical (8) Scheck, R. A.; Francis, M. B. ACS Chem. Biol. 2007, 2, 247–251. Reactivity of Proteins. In The Wiley Encyclopedia of Chemical Biology; (9) Scheck, R. A.; Dedeo, M. T.; Iavarone, A. T.; Francis, M. B. J. Am. Begley, T., Ed.; Wiley-VCH: Weinheim, 2008. Chem. Soc. 2008, 130, 11762–11770. 16812 9 J. AM. CHEM. SOC. 2010, 132, 16812–16817 10.1021/ja105429n 2010 American Chemical Society Highly Reactive Sequences For PLP-Mediated Bioconjugation ARTICLES cysteine alkylation12 as well as C-terminal modification using native chemical ligation (NCL).19,20 PLP-mediated bioconjugation has been used to modify a number of different proteins in applications ranging from surface and polymer attachment21 to the dual modification of viral coat proteins with different chromophores for light harvesting systems.12 Since the initial report of this method for protein functionalization, our group has sought to explore the scope of this reaction and to improve its reliability for new protein targets. Our previous work has shown that the transamination reactivity of a protein is dependent on the identity of the N-terminal residue, as well as those in the second and third positions.9 The terminal and internal residues were found to have a complex synergistic effect on the overall reactivity, quickly outpacing our ability to screen sequence candidates individually. Based on the observation that the penultimate and antepen- ultimate residues are significant in determining the reactivity of peptides and proteins toward PLP-mediated N-terminal Figure 1. The general scheme of PLP-mediated bioconjugation. (a) In the transamination, we sought to identify the most highly reactive first step, a protein is incubated with PLP (1) under mild, aqueous conditions. sequences of amino acids from the chemical space spanning This oxidizes the N-terminus of the protein to a ketone or an aldehyde, all combinations of three N-terminal residues. To identify such providing a unique functional group for further modification. (b) In the sequences, we developed a combinatorial peptide library screen- second step, the ketone is conjugated to an alkoxyamine-bearing reagent (2) through oxime formation. (c) The proposed mechanism begins with ing platform based on a colorimetric detection of desired Schiff base formation between the N-terminal amine and the PLP aldehyde. reactivity, as outlined in Figure 2. Using stringent reaction Tautomerization, followed by hydrolysis, affords the keto-protein product. conditions to screen for the most active sequences, a highly conserved alanine-lysine motif was identified. The improved specificity by targeting the N-terminus, a unique position in the reactivity of this motif was verified on peptide substrates, and sequence. When incubated with pyridoxal 5′-phosphate (PLP), the scope was explored using positional scanning experiments. the N-terminal amine undergoes a transamination reaction that Additionally, we introduced this sequence on two proteins using installs a ketone or an aldehyde in that position without site-directed mutagenesis. In both cases, the alanine-lysine modifying lysine side chain amines.7 The unique reactivity of mutants exhibited improved yields relative to the wild-type the new carbonyl group relative to native side chain function- termini. It is anticipated that this new, highly reactive motif alities allows for further conjugation to alkoxyamine-containing will serve to increase the reactivity of many other protein targets. probes via oxime formation10,11 (Figure 1). This technique provides a convenient and readily scalable way to install a single Results and Discussion functional group in a single location and has been shown to tolerate free cysteine residues.4,5,12 Design of a Peptide Library Screening Platform. Although - the reactivity of full-sized proteins will undoubtedly be influ- There are other methods of N-terminal modification,13 17 enced by their tertiary structure, previous work9 had demon- each of which may be suitable for different applications and strated that general trends in N-terminal reactivity were con- proteins. N-terminal tryptophan residues16 can be ligated to sistent between peptide and protein substrates. This observation aldehyde reagents via a Pictet-Spengler reaction, but it can be suggested that it would be possible to screen peptide libraries difficult to obtain proteins with a tryptophan in the N-terminal to identify sequences that would retain their reactivity in the position.18 N-terminal serines and threonines can be cleaved protein context. To do this, we developed a screening platform using sodium periodate to yield aldehyde functionalities for 17 by adapting a number of traditional techniques used for the further derivitization.