Ion-specific control of the self-assembly dynamics of a nanostructured protein lattice.

Self-assembling proteins offer a potential means of creating nanostructures with complex structure and function. However, using self-assembly to create nanostructures with long-range order whose size is tunable is challenging, because the kinetics and thermodynamics of protein interactions depend sensitively on solution conditions. Here we systematically investigate the impact of varying solution conditions on the self-assembly of SbpA, a surface-layer protein from Lysinibacillus sphaericus that forms two-dimensional nanosheets. Using high-throughput light scattering measurements, we mapped out diagrams that reveal the relative yield of self-assembly of nanosheets over a wide range of concentrations of SbpA and Ca(2+). These diagrams revealed a localized region of optimum yield of nanosheets at intermediate Ca(2+) concentration. Replacement of Mg(2+) or Ba(2+) for Ca(2+) indicates that Ca(2+) acts both as a specific ion that is required to induce self-assembly and as a general divalent cation. In addition, we use competitive titration experiments to find that 5 Ca(2+) bind to SbpA with an affinity of 67.1 ± 0.3 µM. Finally, we show via modeling that nanosheet assembly occurs by growth from a negligibly small critical nucleus. We also chart the dynamics of nanosheet size over a variety of conditions. Our results demonstrate control of the dynamics and size of the self-assembly of a nanostructured lattice, the constituents of which are one of a class of building blocks able to form novel hybrid nanomaterials.

Distribution of Clostridium perfringens isolates from piglets in South Korea.

Clostridium perfringens causes various digestive system disease symptoms in pigs. In the present study, 11 C. perfringens isolates were obtained from diarrheic piglets and 18 from healthy piglets. All of the C. perfringens isolates were shown to be type A using a multiplex PCR assay. The ß2 toxin gene was detected in 27/29 C. perfringens isolates, i.e., 81% (9/11) of diarrheic piglets and 100% (18/18) of healthy piglets, and all of the genes had the same sequence. In conclusion, the ß2 toxin gene of C. perfringens was distributed widely in Korean piglets regardless of the incidence of diarrhea, and there was no clear relationship with enteric disease. A pulsed-field gel electrophoresis analysis of DNA digested using SmaI demonstrated the non-clonal spread of C. perfringens isolates from piglets.

Self-assembly of "S-bilayers", a step toward expanding the dimensionality of S-layer assemblies.

Protein-based assemblies with ordered nanometer-scale features in three dimensions are of interest as functional nanomaterials but are difficult to generate. Here we report that a truncated S-layer protein assembles into stable bilayers, which we characterized using cryogenic-electron microscopy, tomography, and X-ray spectroscopy. We find that emergence of this supermolecular architecture is the outcome of hierarchical processes; the proteins condense in solution to form 2-D crystals, which then stack parallel to one another to create isotropic bilayered assemblies. Within this bilayered structure, registry between lattices in two layers was disclosed, whereas the intrinsic symmetry in each layer was altered. Comparison of these data to images of wild-type SbpA layers on intact cells gave insight into the interactions responsible for bilayer formation. These results establish a platform for engineering S-layer assemblies with 3-D architecture.

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

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.

Multidimensional Assembly of S-Layer Proteins on Mobility-Controlled Polyelectrolyte Multilayers.

Polyelectrolyte multilayers have been vastly utilized as an assembling platform for various biomaterials because of their soft and charged surface characteristics, analogous to biomembrane systems. In particular, polyelectrolyte chains with high self-diffusivity can effectively transfer the surface mobility to the assembling biomolecular species, facilitating the ordered self-assembly. Herein, highly diffusional cationic polyelectrolyte chains of linear polyethylenimine are employed to induce direct binding with negatively charged bacterial surface layer proteins, which eventually lead to large-scale two-dimensional crystals. Notably, at the elevated incubation temperature, a transitory intermediate of one-dimensional chain structure is observed. We reveal that this one-dimensional intermediate is a stable precursor toward two-dimensional crystal arrays.

Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics.

The importance of nonclassical, multistage crystallization pathways is increasingly evident from theoretical studies on colloidal systems and experimental investigations of proteins and biomineral phases. Although theoretical predictions suggest that proteins follow these pathways as a result of fluctuations that create unstable dense-liquid states, microscopic studies indicate these states are long-lived. Using in situ atomic force microscopy to follow 2D assembly of S-layer proteins on supported lipid bilayers, we have obtained a molecular-scale picture of multistage protein crystallization that reveals the importance of conformational transformations in directing the pathway of assembly. We find that monomers with an extended conformation first form a mobile adsorbed phase, from which they condense into amorphous clusters. These clusters undergo a phase transition through S-layer folding into crystalline clusters composed of compact tetramers. Growth then proceeds by formation of new tetramers exclusively at cluster edges, implying tetramer formation is autocatalytic. Analysis of the growth kinetics leads to a quantitative model in which tetramer creation is rate limiting. However, the estimated barrier is much smaller than expected for folding of isolated S-layer proteins, suggesting an energetic rationale for this multistage pathway.

Articular surface area of the coronoid process and radial head in elbow extension: surface ratio in cadavers and a computed tomography study in vivo.

PURPOSE: To quantify the articular surface area ratio of the radial head to the coronoid process to gain a better understanding of the stress distribution across these articulations and possibly to explain the patterns of osteoarthritis that are commonly seen in the elbow. METHODS: Thirty cadaveric elbows were harvested and dissected to allow measurement of the radial head and coronoid process articular surfaces. The articular surface areas were measured using the Image J program (National Institutes of Health, Chicago, IL). Twelve men were recruited for this study, and all received a computed tomography (CT) scan of the elbow. A 3-dimensional image of the proximal radioulnar articular surface was created using volume rendering. All specimens were measured 3 times by 2 observers. RESULTS: In the cadaveric measurements, the mean area of the radial head articular fossa was 247.3 +/- 52.6 mm(2) (mean +/- SD). The mean area of the medial facet of the coronoid process was 232.29 +/- 36.5 mm(2), and the mean area of the lateral facet was 141.9 +/- 33.3 mm(2). The articular surface area ratio of radial head to coronoid process was 1:1.5. In the CT measurement, the mean area of the radial head articular fossa was 258.9 +/- 26.3 mm(2). The mean area of the coronoid process articular surface was 376.9 +/- 37.0 mm(2). The articular surface area ratio of radial head to coronoid process was 1:1.46. CONCLUSIONS: The ratio of articular surface area of radial head to coronoid process is 1:1.51 in cadavers and 1:1.46 using a CT in vivo, which is the reverse of the reported force transmission ratio across the elbow joint.

Stepwise growth of a single polymer chain.

By monitoring the modulation of an ionic current passing through a nanoreactor formed from a protein pore, the step-by-step growth of an individual polymer chain was monitored. The observation of polymer growth at the single-molecule level will be useful for studying the kinetics of chain growth or the movement of polymers under confinement. It might also be used to synthesize "molecular fishing lines" in situ, for applications in stochastic sensing.