Pathway of S-Layer Assembly.

Fig. 1 presents a set of in situ AFM images and height profiles selected from a time sequence that reveals multistage assembly comprised of four distinct processes: (i) adsorption of extended monomers onto the SLB, (ii) condensation into amorphous or liquid-like clusters, (iii) rearrangement and folding into crystalline arrays of tetramers, and (iv) growth by new tetramer formation at edge sites of the crystalline clusters.

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Fig. 1.

(A–F) Series of in situ AFM images and height profiles showing dynamics of S-layer organization into 2D crystals on SLBs. Height profiles were measured along the horizontal black lines in each image and are labeled to denote the heights of the lipid bilayer (LB), adsorbed proteins (APs), amorphous clusters (ACs), and crystalline clusters (CCs). Here, Δt indicates time elapsed since introduction of protein solution (concentration C_P  ≅ 16 μg/mL). By Δt = 15 min (A), S-layer monomers had started to adsorb onto the SLBs. During 15 min ≤ Δt ≤ 70 min, the AP coverage increased and by Δt  ≅ 70 min (B), not only was the surface well covered with APs but ACs of nearly uniform heights had formed. By Δt  ≅ 83 min (C), some of the ACs had begun to show the emergence of internal structure, and by Δt  ≅ 87 min (D), nearly all of ACs in C had transformed into CCs with a tetragonal lattice. The height profiles before and after the transformation (C vs. D) demonstrate that the ACs were slightly taller than the CCs and reflect the resulting periodic structure of the CCs. Detailed volume measurements show that the CCs were also more compact. At later times [e.g., Δt  ≅ 108 min (E) and Δt  ≅ 220 min (F)], each CC continued to grow by consuming available APs near the cluster until growth was physically hindered by neighboring CCs. A highly resolved image from mature CCs (G) revealed the tetrameric arrangement and submolecular details (i.e., loop-like structure) of the four S-layer monomers that comprise each lattice unit.

As Fig. 1A shows, following introduction of protein solution, S-layer monomers and/or small oligomers with an average height of about 2 nm and an extended conformation began to adsorb onto the SLB. As the adsorbed protein (AP) coverage increased, two distinct morphological changes occurred over the time period of ∼15–70 min. First, the SLB became densely populated with APs (height profiles, Fig. 1 B–D). Second, amorphous clusters (ACs) of nearly uniform heights (∼10–12 nm) formed on the SLB (Fig. 1B), and these clusters appeared only after high coverage on the SLB was established. Initially these clusters did not reveal any internal structure nor change their dimensions significantly over a time period of ∼30–80 min. However, gradually internal structure and ordering began to emerge in each of the ACs (Fig. 1C). Fig. 1D shows that, after this extended incubation period, nearly all of the ACs in Fig. 1C transformed into crystalline clusters (CCs) within a short period of about 5–10 min.

The height profiles of the ACs and CCs show that, during the course of this transformation, not only did the individual lattice units emerge (labeled with asterisks in the height profile of Fig. 1D), but the CCs became slightly shorter than their amorphous precursors (9 nm vs. 11 nm). Separate volume measurements of ACs and CCs before and after the transition show that the crystalline phase is about 10% more compact than the amorphous phase (See SI Appendix Figs. 3S-1 and 3S-2). Analysis of cluster size and position vs. time shows there is no correlation between either of these characteristics and our ability to resolve the tetrameric lattice (See SI Appendix Figs. 6S-1 and 6S-2; SI Appendix Table 6S-1). Thus the appearance of order structure is not due to stabilization of cluster position but rather is a result of the amorphous-to-crystalline transformation.

After the transformation was complete, each CC grew by creation of new tetramers at its edges until nearly all of the APs were consumed (Fig. 1 E and F). Fig. 2 follows the transformation and growth process for a single cluster and a detailed picture of the complete adsorption, condensation, transformation, and tetramer-by-tetramer growth stages can be seen in Movie S1. A schematic illustrating the sequence of events leading to formation of crystalline S layers as deduced from the above observations is given in Fig. 3.

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Fig. 2.

Sequential in situ AFM height images and surface plots showing the S-layer adsorption, followed by condensation and phase transition of a single cluster as well as its subsequent growth (C_P  ≅ 16 μg/mL). Here, Δt indicates time elapsed since collection of the image in A. (A) At Δt = 0, monomers or small oligomers in an extended conformation have adsorbed to the SLB. (B) By Δt ∼ 62 min the amorphous nucleus has formed but shows signs of emerging order. Images at C and D show the phase transition from the amorphous C to crystalline D state, at which time each individual lattice unit is clearly discernible. After the transformation [(D) Δt ∼ 70.3 to (H) 116.8 min), the crystal grows by formation of new lattice units at—and only at—unpopulated lattice sites along the perimeter of the crystal. For example, between D and E, newly formed lattice units complete the bottom and third rows of the cluster. Times are (A) 0, (B) 62, (C) 65.8, (D) 70.3, (E) 88.5, (F) 95.5, (G) 102.6, and (H) 116.8 min. (See Movie S1 of the growth process.)

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Fig. 3.

(A) Proposed mechanistic scheme of 2D crystallization of S layer from solution on SLBs. Step I: adsorption of S-layer monomers in extended conformations on the surface, step II: condensation of the amorphous cluster, step III: relaxation to the crystalline nucleus, and step IV: self-catalyzed crystal growth. (B) Animated representation of a single crystal growth preceded by the amorphous-to-crystal transition (S-layer monomers on SLBs shown in light blue; oligomers and newly formed lattice units in pink). Adsorbed monomers of extended conformation (1) condense into an amorphous cluster (2). This amorphous precursor then restructures to form a crystal of folded tetramers (dark blue) composed of 5 lattice units (3). The crystalline cluster then grows by formation of new tetramers preferentially at kink sites (4).

Numerous aspects of the growth process are worth addressing. Each CC continued to grow without interruption as long as it was unhindered by neighboring CCs and there were available APs around the cluster. The AP coverage dropped continually as the CCs grew until, near the end of the process, most of the SLB was devoid of APs. This observation implies that, after the initial coverage had been established, adsorption of new protein onto the SLB from the solution was a much slower process than incorporation of protein into the CCs. Thus growth occurred almost exclusively through surface transport and attachment. (See SI Appendix Fig. 2S-1 for further verification) We indirectly verified that new tetramers did not attach to the clusters directly from solution, but formed from the absorbed monomers on the surface by using dynamic light scattering (DLS) to monitor assembly in bulk solution. The DLS data demonstrate that oligomer formation in solution was much slower than on SLBs (See SI Appendix Fig. 4S-1). In addition, to form crystalline clusters in the absence of SLBs required solution protein concentrations that were 2–3 orders of magnitude larger.

The CCs neither moved nor reorientated relative to one another despite the fluidity of the SLB surface (see SI Appendix Figs. 6S-1–6S-3). This was in stark contrast to the APs; sequential images (shown in Movie S1), reveal a rapidly fluctuating AP distribution. Given that SLBs can be highly fluidic, we cannot say whether APs diffuse on the SLB or move with them, but their mobility is critical for CC growth.

Once nucleation was over, new tetramers appeared exclusively at unpopulated lattice sites around the perimeter of the CCs. No tetramers appeared in the surrounding mobile phase of APs. The frequency of attachment to the CCs was somewhat higher at kink sites where the number of nearest neighbors was maximized. Consequently, newly formed lattice units tended to complete entire rows rather than appear at random edge sites (Fig. 1E). (See Fig. 2 and Movie S1). In addition, detachment of a tetramer from a CC was an extremely rare event. Once a tetramer formed, it remained a permanent part of the crystal. This irreversibility is most readily seen in Movie S1.

Analysis of Assembly Kinetics.

These observations suggest a simple model for the growth phase that leads to a specific prediction of CC size vs. time. The rate of increase in the number of tetramers in a cluster dN_T/dt should be proportional to the number of lattice sites around its perimeter (4L/a), the surface concentration of adsorbed proteins n(t), and the rate coefficient for tetramer formation β. For a square island, this is [1]where L is the island width and a is the lattice parameter. Eq. 1 implies that the CC growth rate should be greater for larger initial cluster size and increase as the clusters grow. But as the APs are consumed and n(t) decreases, the growth rate should approach zero. As shown in SI Appendix Eq. S1-7, n(t) can be expressed in terms of the average island area 〈L(t)²〉 vs. time, the surface density of clusters m, and the initial coverage n₀, all of which can be determined from the experiments. Integrating Eq. 1 with the expression for n(t) gives [2]where N_T,0 is N_T(t) at t = 0 and f(t) is a function that approaches a constant at large t and depends on m, L₀, and n₀. (See SI Appendix Eqs. S1–10 for the detailed expression.) Eq. 2 predicts that larger initial cluster size should correspond to higher growth rate and that the rate should be stronger than linear at short times and eventually decline to zero. Because n₀ should increase with bulk solution concentration C_p, Eq. 2 also predicts that the growth rate will increase with C_p.

Fig. 4A shows the measured dependence of CC size on time for a range of initial cluster sizes at C_p  ≅ 16 μg/mL. Fig. 4B shows the growth curves for CCs of similar initial sizes at three different solution concentrations. The data exhibit all of the features predicted by the model as demonstrated by the curves, which are fits according to Eq. 2.

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Fig. 4.

Growth rates of crystalline clusters. (A) The main plot shows growth of individual clusters for a range of initial sizes N_T,0 (C_p  ≅ 16 μg/mL). It demonstrates that growth rate for an individual cluster depends on its size at t = 0, with larger clusters exhibiting faster growth rates. t = 0 is time when the crystal phase appeared. Dotted lines give the fits to experimental data where the fitting was based on the model described in the text according to Eq. 2. The legend relates the data for each curve to the initial number of lattice units in the crystal nuclei. (B) The second plot presents three growth curves and the corresponding theoretical fits obtained from independent experiments at three different concentrations. Three similar sizes of nuclei were compared to emphasize accelerated growth rate as the concentration was increased.

The fits to the data in Fig. 4 give a value for β/a² of 2.0 × 10^-3 s^-1 at C_p  ≅ 16 μg/mL. As Table 1 shows, the values of β derived from experiments with other protein concentrations are similar (See SI Appendix Tables 1S-1–1S-3). To better understand this parameter, we first assume that, like proteins in solution or the lipids themselves, the adsorbed proteins move by diffusive motion in a viscous media. Then β/a is the product of the diffusive collision rate (D/d), where D is the diffusivity and d is the typical jump distance of a few water molecules, and a Boltzmann factor exp(-E_A/kT) in which the activation energy E_A is associated with the creation of new tetramers. From this analysis we obtain E_A ∼ 51 kJ/mol (Table 1). (For details, see SI Appendix Tables 1S-1, 1S-2, and 1S-3 and SI Appendix Eq. S1-17.) To put this value in context, we compare it to that expected from empirical scaling laws that correlate folding times with protein size and activation energies (21). Based on that law the measured E_A is about 50 kJ/mol lower than would be expected for folding of an isolated S-layer protein and the observed tetramer formation time is smaller by more than 15 orders of magnitude. Though we cannot say a priori whether the SLB itself raises or lowers E_A, this reduction in the folding time and activation barrier is reminiscent of that induced by a potent folding catalyst, such as the Pro region found as part of α-lytic protease, the extracellular bacterial protease, which lowers the folding energy by ∼76 kJ/mol (22). The importance of catalysis in tetramer formation at the edge sites around existing S-layer crystals on SLBs was already clear from the lack of any such events in regions away from the crystals. What this comparison to the scaling laws and the example of α-lytic protease provides is an energetic rationale for the accelerated rate.