Article pubs.acs.org/joc
One-Pot Synthesis of High Molecular Weight Synthetic Heteroprotein Dimers Driven by Charge Complementarity Electrostatic Interactions David Hvasanov,† Ekaterina V. Nam,† Joshua R. Peterson,† Dithepon Pornsaksit,† Jörg Wiedenmann,§ Christopher P. Marquis,‡ and Pall Thordarson*,† †
School of Chemistry, The Australian Centre for Nanomedicine and the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and ‡School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia § National Oceanography Center, University of Southampton, Southampton SO14 3ZH, United Kingdom S Supporting Information *
ABSTRACT: Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide−cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein−protein interactions in chemical biology.
■
fragment (di-scFv),13 a 128 kDa hemoglobin dimer (diHb),14,15 a 34 kDa human interleukin 1 receptor antagonist (di-IL-1ra),16 and a 96 kDa lipase−BSA heteroprotein dimer.17 It is worth noting that Hb has a pI of around 7.1−7.518 close to neutrality, largely eliminating any unfavorable ionic interactions between the two proteins. For di-scFv and di-IL-1ra, the reaction was facilitated by targeting the corresponding protein terminal residues. The di-IL-1ra was synthesized by native chemical ligation (NCL) of N-terminal modified IL-1ra, while di-scFV was synthesized from scFV modified on the C-terminus by a cysteineE-TAG sequence.19 Finally, for the lipase-BSA heteroprotein dimer, the reaction was achieved by an azide− alkyne click reaction. The role and importance of protein charge to probe protein function after amino acid modification has been utilized in the literature, such as the use of protein charge ladders.20,21 However, the exploitation of global protein charge to facilitate dimer bioconjugate synthesis via supramolecular interactions has been neglected. In the above examples, the linkers or spacers used have not provided any additional function to the resulting synthetic protein dimers.
INTRODUCTION Protein−protein interactions and processes are essential for cell functionality and play an important role in biological systems. These processes include signal transduction, gene expression, and enzymatic regulation.1,2 Dimerization often allows for biological specificity due to well-defined protein interactions.3 The ability to control the ubiquitous phenomena of protein dimerization allows scientists to manipulate the regulatory function of organisms and their physical structure.4 Control of protein dimerization using small molecular ligands allows for applications in gene expression,5 signal transduction,6 protein therapeutics7 and tumor therapy.8 Additionally, unnatural synthetic protein dimers might provide unique functions in future sensing or nanoscale devices to name but two examples. Although dimerization of proteins has wide applicability, synthetic dimerization by covalent means using small ligands have been largely limited to low molecular weight enzymes/ peptides (20%). Some notable examples do include a 52 kDa dimer of a monoclonal antibody single-chain © 2014 American Chemical Society
Received: July 28, 2014 Published: September 18, 2014 9594
dx.doi.org/10.1021/jo501713t | J. Org. Chem. 2014, 79, 9594−9602
The Journal of Organic Chemistry
Article
Figure 1. Three proteins used in this study and their key properties. For bovine serum albumin (BSA), the sequence of BSA (ExPASy code: P02769) has been aligned onto the X-ray crystallographic structure of human serum albumin (PDB code: 1AO6). Similarly, the modified Acropora millepora green fluorescent protein (GFP) sequence has been aligned with another Acropora millepora GFP structure (PDB code: 2A46). The sequence alignment was performed using ClustalW2 (http:www.ebi.ac.uk/Tools/clustalw2/). The structure for iso-1 cytochrome c (Cyt c) was used without further modification (PDB code: 1YCC). Negative (red), neutral (white), and positive (blue) electrostatic surface features are presented according to the inserted polarized scale. Images were generated with PyMol (Version 1.3, Schrödinger, LLC) using the APBS plug-in to calculate the electrostatic surface potentical.26 The pI values for BSA29 and Cyt c28 were obtained from the literature, while for GFP it has been estimated by theoretical calculations based on its sequence.27 The number of + or − charges refers to the net whole charges at pH = 7.4 assuming only Lys (+), Arg(+), Glu (−), and Asn (−) are charged at that pH (the N- and C-protein terminus cancel each other out). The target cysteine residues for bioconjugation are colored in green and indicated by an arrow.
Herein, we report the synthesis of protein dimers driven by complementary charge interactions using short-chain commercially available bismaleimide linkers. The linkers used in this study include 1,6-bismaleimidohexane 1, 1,8-bismaleimidodiethylene glycol 2, and 1,11-bismaleimidotriethylene glycol 3, which are inert and neutrally charged spacers, allowing investigation of protein charge complementary effects on synthetic yield. We targeted homo- and heteroprotein dimers incorporating the redox protein iso-1 cytochrome c (Cyt c) from Saccharomyces cerevisiae (yeast), bovine serum albumin (BSA), and a mutant green fluorescent protein derived from Acropora millepora (GFP)22 due to their unique property of possessing only a single free cysteine residue. Moreover, extending our previously reported work on light-activated donor−acceptor bioconjugates,23−25 we also targeted the representative formation of protein-based triad systems, consisting of protein dimers with a charged central synthetic donor 5 or acceptor linker 7. High molecular weight heteroprotein dimers (>80 kDa) were covalently linked using cysteine−maleimide coupling26 with site-specific attachment under benign physiological reaction conditions with up to 30% yield due to charge complementarity and favorable electrostatic attraction.
(II) bis(terpyridine) 5 was synthesized in 27% yield from the previously reported [Ru(4′-(4-aminophenyl)-2,2′:6′2″-terpyridine)2[(PF6)2 431 by a HATU-mediated coupling to 6maleimidocaproic acid. Prior to bioconjugation, 5 was exchanged with chloride salt to increase solubility in aqueous solution and yield. The bis-maleimide functionalized viologen 7 w as syn th esized from 4,4 ′-bipyridinium N ,N-di(propylammonia) hexafluorophosphate 632,33 in modest 2% yield as the main product of this reaction appears to be maleamic acid derivatives of 7 that reduce the yield and make the isolation of 7 difficult. Synthesis of Protein Dimers. Homo- and heteroprotein bioconjugates were synthesized in either a one-or-two-step, one-pot approach using maleimide−thiol chemistry (Scheme 2).26 Maleimide-functionalized spacers allow chemoselective and site-specific attachment to cysteine residues of target proteins under benign conditions (pH 7) via Michael addition. It should be noted that the mutant GFP (27.3 kDa), derived from Acropora millepora, was engineered and expressed to contain only a single free cysteine for functionalization at CYS11922 allowing for single site-specific modification. The Cyt c (12.7 kDa) and BSA (66.7 kDa) were chosen as protein dimer targets due to the complementarity of their net charge, positively and negatively charged at physiological pH, respectively as shown in Figure 1. Additionally, they offer the unique property that each protein has a single free cysteine residue (CYS102 and CYS34),34,35 respectively, allowing for chemoselective functionalization with the target bismaleimides 1, 2, 3, 5, and 7. To show how the interactions of BSA, GFP, and Cyt c affected dimer yields, the formation of homo- and hetero-
■
RESULTS AND DISCUSSION Ionic Properties of Proteins Used for Preparing Dimers. We envisioned that supramolecular ionic interactions might be of considerable importance in the formation of protein dimers. Prior to experimental reaction studies of homoand heteroprotein dimer bioconjugates, the protein surface charges were analyzed using PyMol (version 1.3) to determine whether the charge surrounding cysteine is localized or a global effect plays the key role based on the total number of charged residues in the protein. The GFP and Cyt c proteins have a high positive pI of 8.327 and 10.6,28 respectively, with 2−8 + charges at physiological pH = 7.4. To complement the positively charged GFP and Cyt c protein, BSA was included in this study, which has a pI = 4.729 and approximately 13 − charges at pH = 7.4. All three proteins have a solvent-accessible single cysteine (CYS) residue on their surface. The surface CYSs do not appear to be in particularly negatively or positively charged regions on the calculated30 electrostatic surface of these proteins as shown in Figure 1. Synthesis of Bis-maleimide Donor and Acceptor Spacers 5 and 7. The required donor and acceptor spacers 5 and 7 were prepared with the electron-donating or -accepting ligands ruthenium(II) bis(terpyridine) and 4,4′-bipyridinium, respectively (Scheme 1). Maleimide-functionalized ruthenium9595
dx.doi.org/10.1021/jo501713t | J. Org. Chem. 2014, 79, 9594−9602
The Journal of Organic Chemistry
Article
Scheme 1. Synthesis of Bis-maleimide-Functionalized Spacers 5 and 7
Scheme 2. Generic Conditions for Synthesizing Functionalized Protein Dimer Bioconjugates
Table 1. Yield of Homo- and Heteroprotein Dimers from BSA, GFP, and Cyt c with Spacers 1−3 in 20 mM NaH2PO4/ EDTA, pH 7.0 in 5% CH3CN protein dimers from the structurally simple, commercially available maleimide spacers 1−3 was determined by gel electrophoresis with results as shown in Table 1. Representative gel electrophoresis examples from the synthesis of homo- and heteroprotein dimer bioconjugates using combinations of Cyt c, GFP, and BSA and 1,11bismaleimidotriethylene glycol 3 are shown in Figure 2. As an example, the Cyt c-1-BSA dimer was synthesized via cysteine−maleimide coupling using spacer 1. Acetonitrile was used as a cosolvent to solubilize spacer 1.24 Spacer 1 in a 20 mM phosphate/EDTA-buffered aqueous solution (pH 7) was reacted with reduced Cyt c in 5 fold excess for 2 h prior to addition of BSA (also in 5-fold excess). The first step ensures formation of monofunctionalized Cyt c conjugate, partly due to the CYS102 residue being buried in the hydrophobic pocket of the protein, resulting in slower reaction rates (∼1 h for completion).24 Subsequently, BSA was added with the more reactive cysteine residue (∼2 min for completion)24 exposed in the hydrophilic region of the protein and stirred overnight. A 5fold excess of BSA was added to ensure completion, in part because it has been reported that the actual free cysteine available for functionalization is 0.5 mol of the protein.36 When the results with spacers 1−3 (Table 1) were compared, it was found that only complementary charged
yieldb (%) entry
conjugate
chargea
1
2
3
1 2 3 4 5 6
Cyt c-BSA GFP-BSA Cyt c-Cyt c GFP-Cyt c GFP-GFP BSA-BSA
+/− +/− +/+ +/+ +/+ −/−
24 13
