In vitro self-assembly of tailorable nanotubes from a simple protein building block

Abstract

We demonstrate a method for generating discretely structured protein nanotubes from the simple ring-shaped building block, homohexameric Hcp1 from Pseudomonas aeruginosa. Our design exploited the observation that the crystal lattice of Hcp1 contains rings stacked in a repeating head-to-tail pattern. High-resolution detail of the ring-ring interface allowed the selection of sites for specific cysteine mutations capable of engaging in disulfide bond formation across rings, thereby generating stable Hcp1 nanotubes. Protein nanotubes containing up to 25 subunits ( approximately 100 nm in length) were self-assembled under simple conditions. Furthermore, we demonstrate that the tube ends and interior can be independently and specifically functionalized to generate nanocapsules.

Fig. 1.

Structure-based design of Hcp1 nanotubes. (A) Overview of the Hcp1 hexameric ring structure. The six Hcp1 monomers are colored differently and shown in surface representation. Three monomers are excluded for the “inside” view. Key dimensions of the Hcp1 ring are given in nanometers. The “top” and “bottom” nomenclature is used within the text to highlight the polarity of the Hcp1 ring. Sites of cysteine substitutions used to generate the Hcp1 nanotubes (Arg-157 and Gly-90) are black, and the cysteine substitution site designed to introduce PAMAM-Mal (Q54) reactivity for “plugging” is white (Fig. 4). Table 1 provides a summary of Hcp1 amino acid substitutions and their utility. (B) Section of the Hcp1 honeycomb crystal lattice (PDB ID code 1Y12) viewed perpendicular to the crystallographic z-axis. Individual Hcp1 crystalline tubes are shown in black or gray to aid visualization. (C) Model of an Hcp1 nanotube containing 5 Hcp1_CC ring subunits (gray) and the two capping subunits, G90C (green) and R157C (blue). The sites of each cysteine substitution (black), and the position of the C terminus on capping subunits (red) are indicated.

Fig. 2.

Characterization and optimization of Hcp1 nanotubes. (A) SEC of purified Hcp1_CC under oxidizing (circles) and reducing (diamonds) conditions. The oxidized sample peak eluted at the column void volume (7 ml), whereas the reduced sample peak coeluted with wild-type Hcp1 at 14 ml (wild-type data not shown). (B) Negative stain TEM and single-particle analysis of Hcp1_CC from the void volume fraction of the SEC in (A). (Scale bar, 45 nm.) (Inset) Averaged structures for Hcp1 nanotubes containing 4–7 ring subunits. (Scale bar, 20 nm.) (C) Electron micrographs of representative Hcp1 nanotubes prepared in vitro from completely reduced subunits. (Scale bar, 15 nm.) (D) Length distribution of Hcp1 nanotubes assembled in vivo (white) or in vitro (gray), expressed as the percentage of rings found in tubes of various length intervals. For both assembly methods, the population of tubes of each length (three or more rings) observed in representative transmission electron micrographs was counted (in vivo, n = 190; in vitro, n = 287). The sum of tubes of each length was multiplied by its respective length to yield the number of rings associated with each observed tube length. These subtotals are represented as percentages of the total number of rings observed in all tubes. Tubes with lengths >10 rings were not detected (N/D) in the in vivo assemblies.

Fig. 3.

Chain termination and end differentiation of Hcp1 nanotubes. (A) Nonreducing SDS/PAGE analysis of capping subunits (–) or capping subunits containing various concentrations of Hcp1_CC (total capping subunits:Hcp1_CC, 3:1, 5:4, 2:3, 3:8) and the same total protein concentration. (B) TEM micrographs illustrating single-ended (Left) and double-ended (Right) antibody labeling of Hcp1 nanotubes capped with epitope-tagged subunits.

Fig. 4.

PAMAM-Mal dendrimers react specifically and efficiently with Hcp1 Q54C. (A) SDS/PAGE analysis of Hcp1 Q54C and wild-type Hcp1 (rings in schematic) model reactions with G2 PAMAM-Mal₁₆ (filled sphere in schematic). (B) Optimization of the PAMAM-Mal dendrimer system for reaction with Hcp1 Q54C. Values in parentheses indicate the number of Hcp1 Q54C monomers reacted with dendrimer. (C) PAMAM-Mal reacts inside the central hole of Hcp1 Q54C. TEM micrographs of Hcp1 (control) and Hcp1 Q54C G3-PAMAM-Mal₃₂. (D) Comparison of thiol Sepharose reactivity of Hcp1 wild-type and Hcp1 proteins containing surface (R157C) and inner (Q54C) cysteine substitutions. After incubation, equal quantities of thiol Sepharose bead-associated (B) and unreacted flow-although (F) were analyzed by SDS/PAGE. (E) G3 PAMAM-Mal₁₆ reaction with Hcp1 nanotubes containing the Q54C capping subunits (total capping subunits:Hcp1_CC, 3:2).