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.2012 Aug 7;109(32):12968-73.
doi: 10.1073/pnas.1201504109. Epub 2012 Jul 20.

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

Affiliations

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

Seong-Ho Shin et al. Proc Natl Acad Sci U S A..

Abstract

The concept of a folding funnel with kinetic traps describes folding of individual proteins. Using in situ Atomic Force Microscopy to investigate S-layer assembly on mica, we show this concept is equally valid during self-assembly of proteins into extended matrices. We find the S-layer-on-mica system possesses a kinetic trap associated with conformational differences between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of the monomer phase, however, they then track along two different pathways. One leads directly to the final low-energy state and the other to the kinetic trap. Over time, the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation we find that the energy barriers to formation of the two states differ by only 0.7 kT, but once the high-energy state forms, the barrier to transformation to the low-energy state is 25 kT. Thus the transient state exhibits the characteristics of a kinetic trap in a folding funnel.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) AFM height profile and height image of 2D S-layers assembled on mica showing the two types of domains [incubation temperature (Ti) = 25 °C, protein concentration (CP) = 41 μg/mL in a buffer solution of 10 mM tris(hydroxymethyl)aminomethan (Tris), pH 7.2, 50 mM CaCl2, 100 mM NaCl]. The height profile (Top) measured along the black dotted line in the height image shows the average difference in domain height (Δh) as measured between the blue (tall phase:T) and red (short phase:S) dotted lines is approximately 3 nm. The average height of the tall domains measured from the mica surface (highlighted by white dotted circles) corresponds to ca. 8–9 nm, which is consistent with measurements of S-layers on SLBs. (B–D) Time sequence of in situ AFM images and height profiles showing the early stage of S-layer assembly (CP = 70 μg/mL,Ti = 25 °C). White circles highlight initial nuclei formed from the adsorbed proteins. Height profiles were measured along the horizontal dotted black lines in each image. Blue and red circles in each image highlight a pair of clusters, one tall (blue) and the other short (red). They are denoted in the height profiles by the blue and red stars, respectively, and maintain a consistent height difference of approximately 3 nm during the observed growth period. The nonzero baselines in each height profiles denote the level of the adsorbed monomers, which have an average height of approximately 2 nm. The images were captured at (B) 37, (C) 48, and (D) 59 min after start of the in situ growth experiment.
Fig. 2.
Fig. 2.
Temperature dependence of ratio between number of short and tall domains of S-layer crystals on mica. (A) Relative populations by area fraction of tall (T), short (S), and empty (NC) domains for three different incubation temperatures (Ti). Relative populations depend onTi, with higher temperatures producing larger populations of the more stable tall domains. S-layers were grown atCP = 40 μg/mL andTi = 4, 25 °C, and 37 °C for approximately 4 h and were imaged ex-situ. From these data, the difference between the barriers to formation of the tall and short domains is approximately 1.6 kJ/mol. (SeeSI Appendix for the detailed analysis.) (B) Kinetics of short-to-tall domain transformation. The plot shows the percent area covered by tall domains as a function of time. Labeling of data points (i–iv) corresponds to the image in Fig. 2A on which the analysis was performed. Solid line gives a guide to the eye. Times at which images were captured are (i) 4.5, (ii) 6.8, (iii) 10.0, and (iv) 29.2 h. (C) Plot of relative number ratio of short phase domains (fS) vs. time showing a simple exponential dependence. The best linear fit is shown as a solid line. From these data, the energy barrier to transformation of a domain from short to tall was found to be about approximately 61 kJ/mol (seeSI Appendix for details of the analysis).
Fig. 3.
Fig. 3.
Sequences of in situ AFM images showing transformation from short to tall domains. Δt indicates time elapsed since collection of imagesi andi*. S-layer crystals were initially grown in 5 mL solution at 25 °C and further development of the domains was monitored in the same solution. Series inA shows evolution in distribution of domains in protein solution withCP = 41 μg/mL (10 mM Tris pH 7.2, 50 mM CaCl2, 100 mM NaCl) following an incubation time of 4.5 h. (i) Initial ratio of tall to short domains by area was 1.3. (ii) By Δt ∼ 2.3 h, about 25% of the short domains ini had transformed into tall domains without any dissolution of S-layer proteins from the domains. (iii) and (iv) At Δt ∼ 3.2 and 19.2 h, most of the short domains had transformed into tall domains. However, a few short domains still remained, indicating that the transformation does not go to 100% completion until much later times. Series inB shows transformation of a single domain in protein solution withCP = 70 μg/mL (10 mM Tris pH 7.2, 50 mM CaCl2, 100 mM NaCl) following an incubation time of 2.5 h. The transition began at the free edge of the short domain. In between (iii*) and (iv*), the short domain continued to grow by adding new tetramers at the bottom edge. Times of image collection were (i*) 2.5,(ii*) 2.7, (iii*) 2.8, and (iv*) 2.9 h.
Fig. 4.
Fig. 4.
High-resolution AFM images of tall (A,C) and short (B,D) domains.A andB show unprocessed AFM images.C andD show high resolution height images of four tetramers from tall and short domains that reveal distinct topological differencesC andD were processed through correlation averaging (seeSI Appendix for details). S-layers were initially grown in protein solution withCp ∼ 41 μg/mL (10 mM Tris pH 7.2, 50 mM CaCl2, 100 mM NaCl) at 25 °C for 4.5 h. BothA andB were imaged in the same solution with minimal imaging forces (ca. 85 pN) (full Z color scale: 8 nm forA andB, 2.5 nm forC andD).
Fig. 5.
Fig. 5.
(A) Proposed scheme of multiple pathways of S-layer assembly on mica. S-layer assembly on mica starts from the condensation of monomer S-layers and then follow two different pathways. One leads to low energy, final ordered state known as tall phase (T phase); however, the other leads to kinetically trapped, more disordered state known as short phase (S phase). Early crystal clusters are defined as crystalline domains of S-layers that are still in the process of the growth whereas mature crystal grains are defined as fully grown, closely neighbored crystalline domains of which their growth is limited due to no available space for monomers. (B) Proposed energy diagram of S-layer assembly from the monomer in solution to the crystal on mica. Energy barriers to formation of two states only differ by 1.6 kJ/mol; however, the energy barrier to transformation from one to the other is approximately 38 times larger. These energy barriers are qualitatively presented along with two pathways during the assembly.
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