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Studies of ion channels using expressed protein ligation
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Studies of ion channels using expressed protein ligation Paul J Focke and Francis I Valiyaveetil Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K+ channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review. Address Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, United States Corresponding author: Valiyaveetil, Francis I ([email protected])
Current Opinion in Chemical Biology 2010, 14:797–802 This review comes from a themed issue on Methods for Biomolecular Synthesis and Modification Edited by Matt Francis and Isaac Carrico Available online 19th October 2010 1367-5931/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2010.09.014
Introduction Integral membrane proteins (IMPs) including ion channels, transporters, and receptors play important roles in virtually all aspects of biology. In recent years, there has been a rapid increase in the number of IMP crystal structures [1]. For example, structures of a b-adrenergic receptor [2], glutamate receptor [3], and an inward rectifier K+ channel [4] have been recently reported. This high-resolution structural information has set the stage for incisive structure–function investigations. Structure–function investigations are typically carried out using site directed mutagenesis (SDM). SDM enables substitution of the residue of interest with any of the other naturally occurring amino acids. The limitation of SDM is that the modifications are constrained by the set of naturally occurring amino acids. To overcome these limitations, both nonsense suppression approaches [5] and chemical synthesis [6,7] methods have been developed for the incorporation of unnatural amino acids (UAA). In contrast www.sciencedirect.com
to SDM, UAA mutagenesis allows the introduction of an almost limitless variety of modifications. Through the incorporation of suitable UAAs, the properties of an amino acid side chain can be altered in a systematic manner to precisely define its role in protein structure and function. This review focuses on the use of chemical synthesis methods to incorporate UAAs into ion channels. For recent advances in nonsense suppression methodology, we refer the reader to the following reviews [8,9]. Chemical synthesis enables precise modification of the structural and electronic properties of proteins. The major factor limiting the use of chemical synthesis to investigate proteins has been the size of proteins [10]. Chemical synthesis is carried out using solid phase peptide synthesis protocols (SPPS), which is limited to peptides 50– 60 amino acids in length. This limitation was overcome by the development of the native chemical ligation reaction (NCL) [11,12]. NCL is a reaction between a C-terminal thioester peptide and a peptide with an N-terminal Cys that links the peptides with a native peptide bond at the ligation site. A protein can therefore be assembled from a number of suitably sized peptides by using NCL. NCL can also be used for protein semisynthesis in which a protein is assembled from a synthetic peptide and a protein segment or segments obtained by recombinant means [13]. This version of NCL, referred to as expressed protein ligation (EPL), has greatly simplified the task of engineering proteins and extended the size limits of proteins that can be modified using chemical synthesis [14]. While EPL has found extensive applications in the investigations of soluble proteins [15], in the field of IMPs EPL has been used to investigate ion channels (Table 1). Here, we review recent reports on the applications of EPL in the investigation of ion channels.
Semisynthesis of ion channels: strategies and techniques Semisynthesis of NaK
The NaK channel is a bacterial, non-selective cation channel that conducts Na+, K+, Rb+, and Ca2+ ions [16,17]. The NaK channel structure [18–20] is similar to the pore domain of K+ channels [21–23], but with differences in the selectivity filters. To investigate ion coordination in the selectivity filter of the NaK channel, a semisynthetic strategy was developed that enabled modification of the selectivity filter using chemical synthesis [24] (Figure 1a). Owing to the relatively short size (four identical subunits, 110 amino acids in length), the NaK subunit could be assembled from two peptides. An EPL Current Opinion in Chemical Biology 2010, 14:797–802
798 Methods for Biomolecular Synthesis and Modification
Table 1 Summary of engineered membrane proteins Protein
Size (No. aa)
Phospholamban Influenza A virus M2 HIV virus protein u (Vpu) MscL HCV protease cofactor KcsA OmpF NaK
51 97 81 136 66 123/160 340 91
Description
Method
Refs
SR-reg. protein Proton channel HIV-1 accessory prot. Ion channel Cofactor protein K+-channel Porin Cation channel
Total synthesis Total synthesis Total synthesis Total synthesis Total synthesis Semisynthesis Semisynthesis Semisynthesis
[44] [45] [46] [47] [48] [27,34,40,42,49] [32] [24]
Abbreviations: aa, amino acids; SR-reg. prot., Sarcoplasmic reticulum regulatory protein; HIV, human immunodeficiency virus; MscL, mechanosensitive channel of large conductance; HCV, hepatitis C virus; KcsA, K+ channel from Streptomyces lividans; OmpF, porin from Escherichia coli; NaK, non-selective cation channel from Bacillus cereus.
strategy was used in which the C-terminal peptide (encompassing the selectivity filter) was generated by SPPS while the N-terminal peptide was obtained by recombinant expression. A challenge in the semisynthesis was the purification of the synthetic C-peptide. Purification of the synthetic peptide was accomplished by temporarily introducing Arg residues, an approach that has previously been used for the purification of hydrophobic peptides [25,26]. The NaK N-peptide thioester was obtained recombinantly using a dual-fusion approach
[27]. In the dual-fusion, NaK channel sequences were sandwiched between glutathione-S-transferase (GST), to direct expression of the fusion protein to inclusion bodies [28] and the gyrA intein, to generate the thioester group [29]. Expression in inclusion bodies was used to avoid the cell lethality observed on expression of inteins fused to membrane spanning peptides [27]. The NaK polypeptide was assembled by EPL and folded in vitro to the native state using lipid vesicles. Function-
Figure 1
Semisynthesis of NaK and OmpF. (a) NaK semisynthesis accomplished by EPL of a recombinant thioester (blue) and a synthetic peptide (red). Following assembly, the NaK polypeptide is folded into the native tetrameric state. Two subunits are shown (PDB: 2ahy), with the selectivity filter in stick representation. Structures of the UAAs cysteine sulfonic acid (Csa) and homoserine (Hse), substituted for Asp66, are shown. (b) OmpF semisynthesis accomplished by EPL of a synthetic thioester (red) and a recombinant polypeptide (blue). Following assembly, the OmpF polypeptide is folded into the native trimeric state (one subunit is shown, PDB: 2omf). The site of dansyl fluorophore incorporation is shown in yellow. For panels a and b, the ligation site is colored green. Current Opinion in Chemical Biology 2010, 14:797–802
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Studies of ion channels using expressed protein ligation Focke and Valiyaveetil
Figure 2
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ality of the semisynthetic NaK channel was demonstrated using 86Rb+ flux assays [16]. This semisynthetic strategy was utilized to substitute the native Asp in the NaK selectivity filter with the UAAs homoserine (Hse) and cysteine sulfonic acid (Csa). Interestingly, these investigations revealed a requirement for a negative charge in the selectivity filter of the NaK channel for optimal ion flux. Semisynthesis of OmpF
The outer membrane protein F (OmpF) porin facilitates the translocation of solutes (<600 Da) across the outer membrane of Escherichia coli [30]. OmpF is a trimer in which each subunit (340 amino acids long) has a b-barrel architecture consisting of 16-stranded antiparallel bsheets [31]. Semisynthetic assembly of the OmpF porin was carried out from two segments, a synthetic thioester peptide corresponding to the N-terminal 26 residues and a recombinant fragment corresponding to the rest of the protein with an N-terminal Cys (N-Cys) [32] (Figure 1b). To generate the N-Cys in the recombinant fragment, it was expressed with the Cys preceded by a Met. Following expression, Met was removed by endogenous methionine aminopeptidases to provide the required N-Cys fragment. The N-terminal thioester fragment was obtained by SPPS. A Tyr residue in the synthetic peptide was substituted with propargyltyrosine ether and labeled with the dansyl fluorophore using click chemistry before EPL. The modified thioester peptide was ligated to the recombinant fragment to provide the OmpF polypeptide, which was folded in vitro to the native state. This strategy provided the synthetically modified OmpF porin. This semisynthesis has set the stage for obtaining synthetically modified OmpF porins for biotechnological applications. Modular semisynthesis of KcsA
A limitation of the approach used for the semisynthesis of the NaK channel and the OmpF porin is that it can be used only for relatively small membrane proteins or for the chemical manipulation of the 50 residues at the Nterminus or C-terminus of a protein. To overcome this limitation, a modular strategy was developed [33] that allows chemical synthesis to be used to manipulate any region of the polypeptide chain. The modular approach was applied for the semisynthesis of the full-length KcsA K+ channel (Figure 2a). The previously reported
Semisynthesis of KcsA. (a) Modular strategy. EPL between a recombinantly expressed peptide and a synthetic peptide corresponding to the region of interest (ROI, green) generates an intermediate peptide. Subsequently, the Thz protecting group (red) is removed, and a second EPL reaction with a recombinantly expressed thioester peptide yields the KcsA polypeptide. The polypeptide is folded www.sciencedirect.com
Figure 2 Legend (Continued) in vitro to the native tetrameric state (two subunits are shown, PDB:1k4c). KcsA channels were obtained in which the selectivity filter (left) or the pore helix (right) was obtained by chemical synthesis. Ligation sites are shown in yellow. (b) Close-up view of KcsA selectivity filter and pore helices. Shown are two subunits. The modular strategy was utilized to substitute Trp 68 with 3BT. Semisynthesis was also used to substitute Gly77 with D-Ala and to replace the Tyr78-Gly79 amide peptide bond with an ester. Current Opinion in Chemical Biology 2010, 14:797–802
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semisynthesis only provided a truncated form of KcsA [27,34]. In the modular approach, the KcsA subunit was assembled from three fragments using two EPL reactions. The central fragment encompassing the region of interest (ROI) was obtained by SPPS while the other peptides were obtained by recombinant means. Specifically, the N-terminal thioester peptide was obtained by the dual-fusion approach [27–29], while a sumo-fusion and proteolysis approach [35,36] was developed for generating the C-terminal peptide harboring an N-terminal Cys (N-Cys).
confirming the hypothesis that the D-Ala substitution prevents filter collapse. A functional analysis indicated that the D-Ala mutant, while selective for K+ over Na+, was capable of conducting Na+ ions in the absence of K+ [40]. Similar Na+ conduction in the absence of K+ was not observed for the wild type channel. These results demonstrated that the collapse of the selectivity filter at low K+ is essential for preventing Na+ conduction in the absence of K+.
Assembly of the KcsA polypeptide was performed in the C to N direction. First, the synthetic central fragment was ligated to the recombinant C-terminal peptide to provide an intermediate peptide. In this reaction, the N-Cys of the synthetic peptide was protected with a 1,3-thiazolidine-4-carboxo (Thz) group to prevent cyclization and/or polymerization of the synthetic peptide [37]. Following ligation, this N-Cys was de-protected, allowing for the second ligation step between the N-peptide thioester and the intermediate peptide to generate the KcsA polypeptide. Following assembly, the KcsA polypeptide was folded to the native tetrameric state using lipid vesicles [38]. This modular strategy was utilized to assemble semisynthetic KcsA channels in which the pore helix or the selectivity filter was generated by SPPS. This approach allowed for the selective introduction of the UAA b-(3-benzothienyl)-L-alanine (3-BT) in place of the highly conserved pore helix Trp (Trp68), to investigate H-bond interactions between the pore helix and selectivity filter (Figure 2b). A functional comparison of the 3BT mutant channel to the wild type indicated that the Hbond interactions of Trp68, while not crucial for K+ conduction or selectivity, serve to limit Rb+ conduction through the selectivity filter.
The selectivity filter of K+ channels contains four sequential ion-binding sites that are equivalent in binding K+ [21,41]. These ion-binding sites are formed mainly by the backbone carbonyl oxygen atoms. Owing to the involvement of the protein backbone, semisynthesis rather than SDM was used to manipulate the ionbinding sites in the selectivity filter [5,14]. Semisynthesis was used to substitute the Tyr78 to Gly79 amide bond in the selectivity filter with an ester linkage [42] (Figure 2b). It was predicted that the amide to ester substitution, which reduces the electro-negativity of the carbonyl oxygen [43], would reduce K+ binding at the site. The Tyr78 to Gly79 amide bond was selected as it contributes to only one ion-binding site. The crystal structure of the mutant channel revealed that the ester substitution did not affect the structure of the filter but resulted in reduced binding of K+ at that site. Therefore, in the ester mutant, the binding sites were not equivalent in binding K+. Electrophysiological analysis of the ester mutant showed reduced K+ conduction and an altered conductance-concentration profile. These results indicate that energetic equivalence of the ion-binding sites in the selectivity filter is necessary for optimal K+ flux through the channel.
Applications of semisynthesis in investigations of the selectivity filter of a K+ channel
Conclusion
acid substitution in the selectivity filter of K+ channels D-amino
The selectivity filter of the KcsA channel undergoes a conformational change at low concentrations of K+ to a non-conducting, collapsed state [21,39]. To understand the role of this collapsed state, semisynthesis was used to generate a mutant KcsA channel that does not undergo this conformational change [40]. It was hypothesized that substituting Gly77 with D-Ala would prevent this conformational change, owing to steric clashes of the DAla methyl side chains in the collapsed state (Figure 2b). Semisynthesis was used to generate the Gly77 ! D-Ala mutant and the crystal structure of the D-Ala mutant channel was determined in high and low K+. The structure of the selectivity filter of the D-Ala mutant at high K+ was similar to the structure at low K+. The D-Ala mutant did not undergo a conformational change at low K+, thus Current Opinion in Chemical Biology 2010, 14:797–802
Amide-to-ester substitution in the selectivity filter of K+ channels
The ability to semisynthetically engineer membrane proteins offers great promise toward elucidating the precise molecular mechanisms of ion channels, transporters, receptors, and pores. The successful implementation of the modular semisynthetic approach offers the promise that, with continued effort, more complex IMPs will become amenable to semisynthetic manipulation. We anticipate that the semisynthetic engineering of IMPs will contribute to the elucidation of basic biological questions, and will also find important applications in biotechnology and nanotechnology.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements Research in the Valiyaveetil laboratory is supported by National Institutes of Health (GM087546), and a Scientist Development Grant from the American Heart Association (0835166N). FIV is a Pew Scholar in the Biomedical Sciences. www.sciencedirect.com
Studies of ion channels using expressed protein ligation Focke and Valiyaveetil
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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23. Long SB, Tao X, Campbell EB, MacKinnon R: Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 2007, 450:376-382. 24. Linn KM, Derebe MG, Jiang Y, Valiyaveetil FI: Semisynthesis of NaK, a Na(+) and K(+) conducting ion channel. Biochemistry 2010, 49:4450-4456. The authors describe the semisynthesis of the NaK channel, and use semisynthetic variants of the NaK channel to demonstrate that the acidic residue in the selectivity filter is necessary for optimal ion flux through the channel.
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27. Valiyaveetil F, MacKinnon R, Muir T: Semisynthesis and folding of the potassium channel KcsA. J Am Chem Soc 2002, 124:9113-9120. 28. Frangioni JV, Neel BG: Solubilization and purification of enzymatically active glutathione-S-transferase (pGEX) fusion proteins. Anal Biochem 1993, 210:179-187. 29. Southworth MW, Amaya K, Evans TC, Xu MQ, Perler FB: Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques 1999, 1999:110-114. 30. Schirmer T: General and specific porins from bacterial outer membranes. J Struct Biol 1998, 121:101-109. 31. Cowan SW, Schirmer T, Rummel G, Steiert M, Ghosh R, Pauptit RA, Jansonius JN, Rosenbusch JP: Crystal structures explain functional properties of two E. coli porins. Nature 1992, 358:727-733. 32. Reitz S, Cebi M, Reiss P, Studnik G, Linne U, Koert U, Essen LO: On the function and structure of synthetically modified porins. Angew Chem Int Ed Engl 2009, 48:4853-4857. Semisynthesis and click chemistry is used to introduce a synthetic modulator into the trimeris porin OmpF. The semisynthesis described is likely to be of general use in future protein engineering of porins. 33. Komarov AG, Linn KM, Devereaux JJ, Valiyaveetil FI: Modular strategy for the semisynthesis of a K+ channel: investigating interactions of the pore helix. ACS Chem Biol 2009, 4:10291038. An improved method for semisynthesis of the KcsA channel is described. It is anticipated that this methodology will have future applications toward the protein engineering of integral membrane proteins with multiple membrane spanning segments. 34. Valiyaveetil FI, Sekedat M, Muir TW, MacKinnon R: Semisynthesis of a functional K+ channel. Angew Chem, Int Ed 2004, 43:2504-2507. 35. Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD, Butt TR: SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics 2004, 5:75-86. 36. Zuo X, Li S, Hall J, Mattern M, Tran H, Shoo J, Tan R, Weiss S, Butt T: Enhanced expression and purification of membrane proteins by SUMO fusion in Escherichia coli. J Struct Funct Genomics 2005, 6:103-111. 37. Bang D, Kent SBH: A one-pot total synthesis of crambin. Angew Chem, Int Ed 2004, 43:2534-2538. 38. Valiyaveetil F, Zhou Y, MacKinnon R: Lipids in the structure, folding, and function of the KcsA K+ channel. Biochemistry 2002, 41:10771-10777. 39. Zhou YF, MacKinnon R: The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. J Mol Biol 2003, 333:965-975. Current Opinion in Chemical Biology 2010, 14:797–802
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40. Valiyaveetil FI, Leonetti M, Muir TW, MacKinnon R: Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation. Science 2006, 314:1004-1007. Semisynthesis is used to demonstrate that a conformational change observed in the KcsA selectivity filter is essential for preventing Na+ ion permeation in the absence of K+ ions. 41. Doyle DA, Cabral JM, Pfuetzner RA, Kuo AL, Gulbis JM, Cohen SL, Chait BT, MacKinnon R: The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 1998, 280:69-77. 42. Valiyaveetil F, Sekedat M, MacKinnon R, Muir T: Structural and functional consequences of an amide-to-ester substitution in the selectivity filter of a potassium channel. J Am Chem Soc 2006, 128:11591-11599. This paper highlights the ability of semisynthesis to specifically perturb ion-binding sites in the selectivity filter of the KcsA channel.
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Current Opinion in Chemical Biology 2010, 14:797–802
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Studies of ion channels using expressed protein ligation Paul J Focke and Francis I Valiyaveetil Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K+ channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review. Address Program in Chemical Biology, Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, United States Corresponding author: Valiyaveetil, Francis I ([email protected])
Current Opinion in Chemical Biology 2010, 14:797–802 This review comes from a themed issue on Methods for Biomolecular Synthesis and Modification Edited by Matt Francis and Isaac Carrico Available online 19th October 2010 1367-5931/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2010.09.014
Introduction Integral membrane proteins (IMPs) including ion channels, transporters, and receptors play important roles in virtually all aspects of biology. In recent years, there has been a rapid increase in the number of IMP crystal structures [1]. For example, structures of a b-adrenergic receptor [2], glutamate receptor [3], and an inward rectifier K+ channel [4] have been recently reported. This high-resolution structural information has set the stage for incisive structure–function investigations. Structure–function investigations are typically carried out using site directed mutagenesis (SDM). SDM enables substitution of the residue of interest with any of the other naturally occurring amino acids. The limitation of SDM is that the modifications are constrained by the set of naturally occurring amino acids. To overcome these limitations, both nonsense suppression approaches [5] and chemical synthesis [6,7] methods have been developed for the incorporation of unnatural amino acids (UAA). In contrast www.sciencedirect.com
to SDM, UAA mutagenesis allows the introduction of an almost limitless variety of modifications. Through the incorporation of suitable UAAs, the properties of an amino acid side chain can be altered in a systematic manner to precisely define its role in protein structure and function. This review focuses on the use of chemical synthesis methods to incorporate UAAs into ion channels. For recent advances in nonsense suppression methodology, we refer the reader to the following reviews [8,9]. Chemical synthesis enables precise modification of the structural and electronic properties of proteins. The major factor limiting the use of chemical synthesis to investigate proteins has been the size of proteins [10]. Chemical synthesis is carried out using solid phase peptide synthesis protocols (SPPS), which is limited to peptides 50– 60 amino acids in length. This limitation was overcome by the development of the native chemical ligation reaction (NCL) [11,12]. NCL is a reaction between a C-terminal thioester peptide and a peptide with an N-terminal Cys that links the peptides with a native peptide bond at the ligation site. A protein can therefore be assembled from a number of suitably sized peptides by using NCL. NCL can also be used for protein semisynthesis in which a protein is assembled from a synthetic peptide and a protein segment or segments obtained by recombinant means [13]. This version of NCL, referred to as expressed protein ligation (EPL), has greatly simplified the task of engineering proteins and extended the size limits of proteins that can be modified using chemical synthesis [14]. While EPL has found extensive applications in the investigations of soluble proteins [15], in the field of IMPs EPL has been used to investigate ion channels (Table 1). Here, we review recent reports on the applications of EPL in the investigation of ion channels.
Semisynthesis of ion channels: strategies and techniques Semisynthesis of NaK
The NaK channel is a bacterial, non-selective cation channel that conducts Na+, K+, Rb+, and Ca2+ ions [16,17]. The NaK channel structure [18–20] is similar to the pore domain of K+ channels [21–23], but with differences in the selectivity filters. To investigate ion coordination in the selectivity filter of the NaK channel, a semisynthetic strategy was developed that enabled modification of the selectivity filter using chemical synthesis [24] (Figure 1a). Owing to the relatively short size (four identical subunits, 110 amino acids in length), the NaK subunit could be assembled from two peptides. An EPL Current Opinion in Chemical Biology 2010, 14:797–802
798 Methods for Biomolecular Synthesis and Modification
Table 1 Summary of engineered membrane proteins Protein
Size (No. aa)
Phospholamban Influenza A virus M2 HIV virus protein u (Vpu) MscL HCV protease cofactor KcsA OmpF NaK
51 97 81 136 66 123/160 340 91
Description
Method
Refs
SR-reg. protein Proton channel HIV-1 accessory prot. Ion channel Cofactor protein K+-channel Porin Cation channel
Total synthesis Total synthesis Total synthesis Total synthesis Total synthesis Semisynthesis Semisynthesis Semisynthesis
[44] [45] [46] [47] [48] [27,34,40,42,49] [32] [24]
Abbreviations: aa, amino acids; SR-reg. prot., Sarcoplasmic reticulum regulatory protein; HIV, human immunodeficiency virus; MscL, mechanosensitive channel of large conductance; HCV, hepatitis C virus; KcsA, K+ channel from Streptomyces lividans; OmpF, porin from Escherichia coli; NaK, non-selective cation channel from Bacillus cereus.
strategy was used in which the C-terminal peptide (encompassing the selectivity filter) was generated by SPPS while the N-terminal peptide was obtained by recombinant expression. A challenge in the semisynthesis was the purification of the synthetic C-peptide. Purification of the synthetic peptide was accomplished by temporarily introducing Arg residues, an approach that has previously been used for the purification of hydrophobic peptides [25,26]. The NaK N-peptide thioester was obtained recombinantly using a dual-fusion approach
[27]. In the dual-fusion, NaK channel sequences were sandwiched between glutathione-S-transferase (GST), to direct expression of the fusion protein to inclusion bodies [28] and the gyrA intein, to generate the thioester group [29]. Expression in inclusion bodies was used to avoid the cell lethality observed on expression of inteins fused to membrane spanning peptides [27]. The NaK polypeptide was assembled by EPL and folded in vitro to the native state using lipid vesicles. Function-
Figure 1
Semisynthesis of NaK and OmpF. (a) NaK semisynthesis accomplished by EPL of a recombinant thioester (blue) and a synthetic peptide (red). Following assembly, the NaK polypeptide is folded into the native tetrameric state. Two subunits are shown (PDB: 2ahy), with the selectivity filter in stick representation. Structures of the UAAs cysteine sulfonic acid (Csa) and homoserine (Hse), substituted for Asp66, are shown. (b) OmpF semisynthesis accomplished by EPL of a synthetic thioester (red) and a recombinant polypeptide (blue). Following assembly, the OmpF polypeptide is folded into the native trimeric state (one subunit is shown, PDB: 2omf). The site of dansyl fluorophore incorporation is shown in yellow. For panels a and b, the ligation site is colored green. Current Opinion in Chemical Biology 2010, 14:797–802
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Studies of ion channels using expressed protein ligation Focke and Valiyaveetil
Figure 2
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ality of the semisynthetic NaK channel was demonstrated using 86Rb+ flux assays [16]. This semisynthetic strategy was utilized to substitute the native Asp in the NaK selectivity filter with the UAAs homoserine (Hse) and cysteine sulfonic acid (Csa). Interestingly, these investigations revealed a requirement for a negative charge in the selectivity filter of the NaK channel for optimal ion flux. Semisynthesis of OmpF
The outer membrane protein F (OmpF) porin facilitates the translocation of solutes (<600 Da) across the outer membrane of Escherichia coli [30]. OmpF is a trimer in which each subunit (340 amino acids long) has a b-barrel architecture consisting of 16-stranded antiparallel bsheets [31]. Semisynthetic assembly of the OmpF porin was carried out from two segments, a synthetic thioester peptide corresponding to the N-terminal 26 residues and a recombinant fragment corresponding to the rest of the protein with an N-terminal Cys (N-Cys) [32] (Figure 1b). To generate the N-Cys in the recombinant fragment, it was expressed with the Cys preceded by a Met. Following expression, Met was removed by endogenous methionine aminopeptidases to provide the required N-Cys fragment. The N-terminal thioester fragment was obtained by SPPS. A Tyr residue in the synthetic peptide was substituted with propargyltyrosine ether and labeled with the dansyl fluorophore using click chemistry before EPL. The modified thioester peptide was ligated to the recombinant fragment to provide the OmpF polypeptide, which was folded in vitro to the native state. This strategy provided the synthetically modified OmpF porin. This semisynthesis has set the stage for obtaining synthetically modified OmpF porins for biotechnological applications. Modular semisynthesis of KcsA
A limitation of the approach used for the semisynthesis of the NaK channel and the OmpF porin is that it can be used only for relatively small membrane proteins or for the chemical manipulation of the 50 residues at the Nterminus or C-terminus of a protein. To overcome this limitation, a modular strategy was developed [33] that allows chemical synthesis to be used to manipulate any region of the polypeptide chain. The modular approach was applied for the semisynthesis of the full-length KcsA K+ channel (Figure 2a). The previously reported
Semisynthesis of KcsA. (a) Modular strategy. EPL between a recombinantly expressed peptide and a synthetic peptide corresponding to the region of interest (ROI, green) generates an intermediate peptide. Subsequently, the Thz protecting group (red) is removed, and a second EPL reaction with a recombinantly expressed thioester peptide yields the KcsA polypeptide. The polypeptide is folded www.sciencedirect.com
Figure 2 Legend (Continued) in vitro to the native tetrameric state (two subunits are shown, PDB:1k4c). KcsA channels were obtained in which the selectivity filter (left) or the pore helix (right) was obtained by chemical synthesis. Ligation sites are shown in yellow. (b) Close-up view of KcsA selectivity filter and pore helices. Shown are two subunits. The modular strategy was utilized to substitute Trp 68 with 3BT. Semisynthesis was also used to substitute Gly77 with D-Ala and to replace the Tyr78-Gly79 amide peptide bond with an ester. Current Opinion in Chemical Biology 2010, 14:797–802
800 Methods for Biomolecular Synthesis and Modification
semisynthesis only provided a truncated form of KcsA [27,34]. In the modular approach, the KcsA subunit was assembled from three fragments using two EPL reactions. The central fragment encompassing the region of interest (ROI) was obtained by SPPS while the other peptides were obtained by recombinant means. Specifically, the N-terminal thioester peptide was obtained by the dual-fusion approach [27–29], while a sumo-fusion and proteolysis approach [35,36] was developed for generating the C-terminal peptide harboring an N-terminal Cys (N-Cys).
confirming the hypothesis that the D-Ala substitution prevents filter collapse. A functional analysis indicated that the D-Ala mutant, while selective for K+ over Na+, was capable of conducting Na+ ions in the absence of K+ [40]. Similar Na+ conduction in the absence of K+ was not observed for the wild type channel. These results demonstrated that the collapse of the selectivity filter at low K+ is essential for preventing Na+ conduction in the absence of K+.
Assembly of the KcsA polypeptide was performed in the C to N direction. First, the synthetic central fragment was ligated to the recombinant C-terminal peptide to provide an intermediate peptide. In this reaction, the N-Cys of the synthetic peptide was protected with a 1,3-thiazolidine-4-carboxo (Thz) group to prevent cyclization and/or polymerization of the synthetic peptide [37]. Following ligation, this N-Cys was de-protected, allowing for the second ligation step between the N-peptide thioester and the intermediate peptide to generate the KcsA polypeptide. Following assembly, the KcsA polypeptide was folded to the native tetrameric state using lipid vesicles [38]. This modular strategy was utilized to assemble semisynthetic KcsA channels in which the pore helix or the selectivity filter was generated by SPPS. This approach allowed for the selective introduction of the UAA b-(3-benzothienyl)-L-alanine (3-BT) in place of the highly conserved pore helix Trp (Trp68), to investigate H-bond interactions between the pore helix and selectivity filter (Figure 2b). A functional comparison of the 3BT mutant channel to the wild type indicated that the Hbond interactions of Trp68, while not crucial for K+ conduction or selectivity, serve to limit Rb+ conduction through the selectivity filter.
The selectivity filter of K+ channels contains four sequential ion-binding sites that are equivalent in binding K+ [21,41]. These ion-binding sites are formed mainly by the backbone carbonyl oxygen atoms. Owing to the involvement of the protein backbone, semisynthesis rather than SDM was used to manipulate the ionbinding sites in the selectivity filter [5,14]. Semisynthesis was used to substitute the Tyr78 to Gly79 amide bond in the selectivity filter with an ester linkage [42] (Figure 2b). It was predicted that the amide to ester substitution, which reduces the electro-negativity of the carbonyl oxygen [43], would reduce K+ binding at the site. The Tyr78 to Gly79 amide bond was selected as it contributes to only one ion-binding site. The crystal structure of the mutant channel revealed that the ester substitution did not affect the structure of the filter but resulted in reduced binding of K+ at that site. Therefore, in the ester mutant, the binding sites were not equivalent in binding K+. Electrophysiological analysis of the ester mutant showed reduced K+ conduction and an altered conductance-concentration profile. These results indicate that energetic equivalence of the ion-binding sites in the selectivity filter is necessary for optimal K+ flux through the channel.
Applications of semisynthesis in investigations of the selectivity filter of a K+ channel
Conclusion
acid substitution in the selectivity filter of K+ channels D-amino
The selectivity filter of the KcsA channel undergoes a conformational change at low concentrations of K+ to a non-conducting, collapsed state [21,39]. To understand the role of this collapsed state, semisynthesis was used to generate a mutant KcsA channel that does not undergo this conformational change [40]. It was hypothesized that substituting Gly77 with D-Ala would prevent this conformational change, owing to steric clashes of the DAla methyl side chains in the collapsed state (Figure 2b). Semisynthesis was used to generate the Gly77 ! D-Ala mutant and the crystal structure of the D-Ala mutant channel was determined in high and low K+. The structure of the selectivity filter of the D-Ala mutant at high K+ was similar to the structure at low K+. The D-Ala mutant did not undergo a conformational change at low K+, thus Current Opinion in Chemical Biology 2010, 14:797–802
Amide-to-ester substitution in the selectivity filter of K+ channels
The ability to semisynthetically engineer membrane proteins offers great promise toward elucidating the precise molecular mechanisms of ion channels, transporters, receptors, and pores. The successful implementation of the modular semisynthetic approach offers the promise that, with continued effort, more complex IMPs will become amenable to semisynthetic manipulation. We anticipate that the semisynthetic engineering of IMPs will contribute to the elucidation of basic biological questions, and will also find important applications in biotechnology and nanotechnology.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements Research in the Valiyaveetil laboratory is supported by National Institutes of Health (GM087546), and a Scientist Development Grant from the American Heart Association (0835166N). FIV is a Pew Scholar in the Biomedical Sciences. www.sciencedirect.com
Studies of ion channels using expressed protein ligation Focke and Valiyaveetil
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