Controlled formation of complex nanostructures is one of the main goals of nanoscience and nanotechnology. Stable Protein 1 (SP1) is a boiling-stable ring protein complex, 11 nm in diameter, which self-assembles from 12 identical monomers. SP1 can be utilized to form large ordered arrays; it can be easily modified by genetic engineering to produce various mutants; it is also capable of binding gold nanoparticles (GNPs) and thus forming protein-GNP chains made of alternating SP1s and GNPs. We report the formation and the protocols leading to the formation of those nanostructures and their characterization by transmission electron microscopy, atomic force microscopy, and electrostatic force microscopy. Further control over the GNP interdistances within the protein-GNP chains may lead to the formation of nanowires and structures that may be useful for nanoelectronics.

We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stressrelated proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization timeof-flight mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. T m of 107°C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 Å resolution by multi-wavelength anomalous dispersion method from a crystal belonging to the space group I422. The phases were extended to 1.8 Å resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-likeshaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.

Stable protein 1 (SP1) is a homo-oligomeric protein isolated from aspen (Populus tremula aspen) plants which forms a ring-shape dodecameric particle with a central cavity. The oligomeric form of SP1 is an exceptionally stable structure that is resistant to proteases (e.g., trypsin, V8, and proteinase K), high temperatures, organic solvents, and high levels of ionic detergent. Analytical ultra-centrifugation, chemical cross-linking, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and transmission electron microscopy were used to further characterize the SP1 dodecamer. Introduction of a single cysteine at the N-terminus of SP1 enabled the formation of disulfide bridges within the SP1 dodecamer, concurrent with increased melting point. A six-histidine tag was introduced at the N-terminus of SP1 to generate 6HSP1, and the DeltaNSP1 mutant was generated by a deletion of amino acids 2-6 at the N-terminus. Both 6HSP1 and DeltaNSP1 maintained their ability to assemble a stable dodecamer. Remarkably, these SP1 homo-dodecamers were able to re-assemble into stable hetero-dodecamers following co-electro-elution from SDS-PAGE. The exceptional stability of the SP1-nano ring and its ability to self-assemble hetero-complexes paves the way to further research in utilizing this unique protein in nano-biotechnology.

The purpose of this study was to investigate various growth parameters, dry matter and nitrogen, phosphorus and potassium allocation and photosynthesis ofCarex acutiformis, C. rostrata andC. diandra growing in fens with, in this order, decreasing nutrient availability and decreasing aboveground productivity. Plants were grown from cuttings at optimum nutrient conditions in a growth chamber. Growth analysis at sequential harvests revealed that the species had no inherently different relative growth rates which could explain their different productivity, but that their LAR (LWR and SLA) decreased in the orderC. acutiformis, C. rostrata, C. diandra and their NAR increased in this order. All growth parameters decreased during plant growth even under the controlled conditions of the experiment.C. acutiformis allocated relatively much dry matter to the leaves,C. rostrata to the rhizomes andC. diandra to the roots. This may, in part, explain the higher aboveground biomass production ofC. acutiformis in the field. Nitrogen, but not phosphorus and potassium, allocation patterns were different for the three species.C. diandra, the species from the nitrogen-poorest site, had the highest leaf N content of the three species and also a higher chlorophyll content. Related to this, this species had the highest photosynthetic activity of whole plants both when collected from the field and when grown in the growth chamber. The nitrogen productivity was similar for the three species and the photosynthetic nitrogen use efficiency, determined forC. acutiformis andC. diandra, was similar for these two species.C. diandra had the most finely branched root system, i.e., the highest specific root length of the three species and its root surface area to leaf surface area ratio was also the highest. All three species showed higher nitrate reductase activity in the leaves than in the roots when grown on nutrient solution. The growth ofC. diandra at a relatively nutrient-poor site and a rather open low vegetation is assumed to be adapted to its habitat by a relatively high NAR made possible by a high rate of photosynthesis concurrent with a high leaf N content. The growth ofC. acutiformis at a relatively nutrient-rich site and a more dense and higher vegetation is adapted to its habitat by a high LAR.