Controlling the Assembly of Coiled–Coil Peptide Nanotubes

Abstract An ability to control the assembly of peptide nanotubes (PNTs) would provide biomaterials for applications in nanotechnology and synthetic biology. Recently, we presented a modular design for PNTs using α‐helical barrels with tunable internal cavities as building blocks. These first‐generation designs thicken beyond single PNTs. Herein we describe strategies for controlling this lateral association, and also for the longitudinal assembly. We show that PNT thickening is pH sensitive, and can be reversed under acidic conditions. Based on this, repulsive charge interactions are engineered into the building blocks leading to the assembly of single PNTs at neutral pH. The building blocks are modified further to produce covalently linked PNTs via native chemical ligation, rendering ca. 100 nm‐long nanotubes. Finally, we show that small molecules can be sequestered within the interior lumens of single PNTs.

Self-assembling peptide-based materials have become progressively more established in biomedicine and nanotechnology,w ith potential applications as matrices for tissue-engineering,drug-delivery systems,and templates for mineralization and metalation. [1] Owing to their large internal surface areas,p eptide nanotubes (PNTs) potentially expand the possible applications to filtration and storage devices,sensors, or even catalysts. [2] To date,P NT designs have used Fmocdipeptides, [3] cyclic b-sheet stacking peptides, [4] lock-washer ahelical bundles as the building blocks, [5] or short peptides that self-assemble into spiral tapes. [6] Recently,w ep resented ageneric modular approach to assemble PNTs from a-helical barrels (aHBs), Figure 1. [7] Theapplication of peptide-based materials requires good control over self-assembly and material properties.F or PNTs this is currently best achieved by self-organizing systems based on cyclic b-sheet stacking peptides. [8] In these cases,the inner diameter can be tailored via the ring size of the peptide. [2] Themodular approach that we present to assemble PNTs from aHBs allows similar control mechanisms. [7a] We have shown that the inner diameter varies with the oligomer state of the aHB,allowing PNTs with channels of 5-7 tobe achieved. However,a ll of these PNTs assemble into broad- Figure 1. Schematic representationf or PNT assembly.a)Self-assembly of broadened PNTsbased on CC-Hex-T; b) self-assembly of single PNTsbased on CC-Hex-T + ;c )covalent assembly of CC-Hex-T + co, where Xand Yrepresent the ligatable thiol and thioester groups (in (a) and (b) red:p ositively charged N-terminus, blue:n egatively charged C-terminus).S equences for CC-Hex-T,C C-Hex-T + ,a nd CC-Hex-T + co, are given in Table 1. ened fibers,F igure 1a.C ontrolling this broadening would represent afurther step towards functional aHB-based PNTs. Herein we describe strategies for this,which include the use of pH and sequence redesigns to make single PNTs,F igure 1b. We compare spontaneous and covalent assembly of the PNTs, Figure 1b,c, and, we show that different PNTs can discriminate in the encapsulation of small molecules.
Our first-generation PNT designs use designed bluntended aHBs. [7c] To promote end-to-end self-assembly,t hese are permuted to expose hydrophobic patches at the Ntermini, and leave overall and complementary negative and positive charges at the C-a nd N-termini, respectively,F igure 1. [7a] As mentioned, these redesigns associated both longitudinally and laterally to give broadened assemblies compared to the widths of the building blocks (Figure 2a,b). To address this herein, we focus on further redesign of the hexameric building block, CC-Hex-T.T his is well-characterized with a6channel that is stable to certain mutations. [7b, 10] Initially,w ei nvestigated the pH-dependence of lateral assembly.W eposited that fiber broadening of CC-Hex-based nanotubes should be pH sensitive as protonation of the glutamate (E) residues at low pH would leave the building blocks with a + 30 charge from the 5lysine residues (K) in the sequence,T able 1. As predicted, at low pH thin fibers were observed ( Figure 2). Moreover,f ibrils consistent with single PNTs (ca. 3-4 nm, Table S1 in the Supporting Information) were observed by negative-stain transmission electron microscopy (TEM) below pH 5.6 ( Figure 2c). Circular dichroism (CD) spectroscopy confirmed this in solution. Owing to chiral scattering,broadened fibrous a-helical systems,such as CC-Hex-T at pH 7.4, give red-shifted CD spectra of reduced intensity,F igure 2b,c ompared with typical a-helical spectra. [11] However,d ecreasing the pH for CC-Hex-T samples gave increased signal and loss of the red shift, with the transition complete by pH 5.6 ( Figure 2d). This disassembly of the fibers,b ut not of the a-helical structure,i na cidic conditions was reversible and thickened fibers returned upon increasing pH ( Figure S2).
Theo bservation of reduced fiber thickening at low pH suggested ar edesign of CC-Hex-T to make single PNTs at neutral pH. We reasoned that increasing the positive charge on the outer surfaces of the fibrils should prevent bundling to form fibers, Figure 1b.Incoiled-coil structures,the f positions of the underlying sequence repeat, abcdefg (Table 1), fall on this outer surface. [12] ForCC-Hex-T + ,wemutated all but one of these to Kg iving an overall positive charge of + 3p er peptide,and + 18 per CC-Hex building block, at neutral pH, Table 1.
When equilibrated at pH 7.4, CC-Hex-T + showed exclusively extended fibrils up to about 1micron in length in TEM, with diameters of around 3-4 nm (Figures 2e and Table S2), that is,c onsistent with single PNTs;a nd without chiral scattering in the CD spectra, Figure 2f.
To improve stability of the single PNT fibrils,w e attempted to link the CC-Hex-T building blocks covalently through native chemical ligation (NCL), Figure 1c. [13] To enable this,wemade athird peptide,CC-Hex-T + co,inwhich CC-Hex-T + was modified to include an N-terminal cysteine (C) and a C-terminal thioester ( Table 1, Scheme 1).
CD spectra before NCL showed stably folded a-helices (Figure 2h). [10] Thus,w ea ssume that polymerization occurs mostly via assembled CC-Hex-based building blocks.A fter 1day of reaction, high-molecular-weight species appeared in Table 1: Sequences of designed PNT-formingp eptides.

CC-Hex-T [a] H-LKAIAQE LKAIAKE LKAIAWE LKAIAQE-OH CC-Hex-T + [a] H-LKAIAKE LKAIAKE LKAIAWE LKAIAKE-OH CC-Hex-T + co [a] H-CKAIAKE LKAIAYE LKAIAKE LKAIAKQ-SBzl
[a] The nomenclature is based on the oligomeric state of the monomer building block, which is acoiled-coil hexamer.  (c) and (e) point out thin fibrils (single PNTs) that are otherwise difficult to see. CD spectra for b) CC-Hex-T; d) CC-Hex-TatpH7.4, 6.7, 6.0, 5.6, 5.1, and 3.1 (black-to-gray gradient:f rom pH 7.4 (black) to pH 3.1 (light gray));f)CC-Hex-T + ; and h) CC-Hex-T + co monomer building block. To explore the timeframe of oligomerization, we monitored the reaction by high-pressure liquid chromatography (HPLC), SDS-PAGE, TEM, and mass spectrometry (MS). HPLC showed over 80 %c onversion of the starting material after 5h ( Figure S5). This was confirmed by SDS-PAGE ( Figure 3g): after 1day,t he monomer band was faint;t he appearance and disappearance of ab and consistent with dimeric non-self-assembling CC-Hex-T + co was visible (Figure S6);a nd ah igh-molecular-weight smear formed over time.M Sr evealed dimers to octamers after three hours (Figures 3h and Figure S7). However,s mall amounts of dimeric and trimeric cyclic species were also present. Corroborating this,T EM images revealed short fibrils of 30-40 nm in length after 30 min, with the average fiber length increasing up to 100 nm after 7days (Figure 3a-f,F igure S8,S9). We suggest that the observation of al imiting length is due to many nucleation sites,and the precipitation of the covalent PNTs from solution.
We turned to linear dichroism (LD) spectroscopy to verify the secondary and quaternary structure of the PNTs in solution. [14a] This requires the alignment of molecules,usually by shear flow,and only gives signal for those with large aspect ratios.W eo bserved LD signals for both heat-treated CC-Hex-T at low pH-which gave longer fibrils than untreated samples,F igure S9-and CC-Hex-T + at neutral pH, Figure 4a,b.T he resulting spectra indicated aligned a-helical rods with the helices parallel to the long axis of the rods. [14] Moreover,t hese correlated with fibril lengths observed in TEM:t he longer CC-Hex-T + fibrils gave the more intense LD signal, and required lower alignment forces,F igure 4b, than the heated CC-Hex-T fibrils,F igure 4a.Incontrast, any LD signals for spontaneously assembled CC-Hex-T,a nd for covalently linked CC-Hex-T + co were too weak to be observed;p resumably,t his reflects relatively short fibers formed by these systems,w hich were 100 nm and at the limit of the size required for LD spectroscopy,F igure S8-S10. [14] Finally,toprobe the accessibilities of the inner channels of the single PNTs,a nd to assess their utility as components of sequestration, storage,a nd delivery devices,w et ested the encapsulation of the small hydrophobic dye 1,6-diphenylhexatriene (DPH). In hydrophobic environments,D PH fluores-

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Chemie ces,F igure 4c,b ut this is quenched in water. DPH binds strongly to thickened CC-Hex-T PNTs (Figure 4d). [7a] Although DPH bound to both of the single-PNT redesigns, Figure 4c,t he saturation binding curve for CC-Hex-T + showed reduced binding affinity compared to CC-Hex-T, Figure 4d.W epropose that this is best explained by CC-Hex-T + PNTs being less stable than CC-Hex-T,a sa ny stabilization from lateral association of fibrils will be lost in the CC-Hex-T + PNTs.Inturn, this facilitates release of bound DPH. Supporting this idea, the covalently linked PNTs of CC-Hex-T + co showed binding behavior similar to CC-Hex-T,F igure 4c,d. We suggest that covalent linkage of the monomers into the extended tubular structures prevents PNT disassembly,a nd, therefore,i ncreases the binding affinity to small hydrophobic molecules.
In summary,wehave demonstrated alternative strategies to control the lateral and longitudinal assembly of a-helical PNTs.T hese systems range from non-covalent assembliesnamely,stable thickened PNTs,which unbundle under acidic conditions;and more-dynamic single PNTs achieved through rational redesign-to highly stable covalently linked single PNTs.T he different assembly modes alter both the morphologies of the PNTs,and their properties,including the uptake and release of hydrophobic molecules.I nf uture,t his could guide the design of PNT-based delivery systems or storage devices.
There are as mall number of other PNT systems that display elements of control over assembly that we demonstrate herein. These include cyclic b-structured peptides that stack, [8] hydrophobic dipeptide-based PNTs, [15] and, in part, the self-assembling spiral tapes based on disulfide-linked octapeptides. [16] Our designs carry certain advantages,principally modularity and generality.T herefore,t he control mechanisms that we describe should be readily transferrable to other aHB building blocks of varying pore size. [7a,c] The inner lumens of these barrels are also mutable, [7b, 10] which should facilitate further the design of functional PNTs.