Solving the Riddle of the Evolution of Shine-Dalgarno Based Translation in Chloroplasts.

Chloroplasts originated from an ancient cyanobacterium and still harbor a bacterial-like genome. However, the centrality of Shine-Dalgarno ribosome binding, which predominantly regulates proteobacterial translation initiation, is significantly decreased in chloroplasts. As plastid ribosomal RNA anti-Shine-Dalgarno elements are similar to their bacterial counterparts, these sites alone cannot explain this decline. By computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine-Dalgarno locus, which in-turn reduce the probability of ribosome binding. To validate our model, we expressed two reporter genes (mCherry, hydrogenase) harboring a Shine-Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. Coexpressing them with a 16S ribosomal RNA, modified according to our model, significantly enhances mCherry and hydrogenase expression compared with coexpression with an endogenous 16S gene.

Binding of ferredoxin to algal photosystem I involves a single binding site and is composed of two thermodynamically distinct events.

Despite the impressive progress made in recent years in understanding the early steps in charge separation within the photosynthetic reaction centers, our knowledge of how ferredoxin (Fd) interacts with the acceptor side of photosystem I (PSI) is not as well developed. Fd accepts electrons after transiently docking to a binding site on the acceptor side of PSI. However, the exact location, as well as the stoichiometry, of this binding have been a matter of debate for more than two decades. Here, using Isothermal Titration Calorimetry (ITC) and purified components from wild type and mutant strains of the green algae Chlamydomonas reinhardtii we show that PSI has a single binding site for Fd, and that the association consists of two distinct binding events, each with a specific association constant.

Disruption of diphenylalanine assembly by a Boc-modified variant.

Peptide-based biomaterials are key to the future of diagnostics and therapy, promoting applications such as tissue scaffolds and drug delivery vehicles. To realise the full potential of the peptide systems, control and optimisation of material properties are essential. Here we investigated the co-assembly of the minimal amyloid motif peptide, diphenylalanine (FF), and its tert-butoxycarbonyl (Boc)-modified derivative. Using Atomic Force Microscopy, we demonstrated that the co-assembled fibers are less rigid and show a curvier morphology. We propose that the Boc-modification of FF disrupts the hydrogen bond packing of adjacent N-termini, as supported by Fourier transform infrared and fluorescence spectroscopic data. Such rationally modified co-assemblies offer chemical functionality for after-assembly modification and controllable surface properties for tissue engineering scaffolds, along with tunable morphological vs. mechanical properties.

Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I.

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 µM and ∆H of -20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ∆CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Å2. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.

Controlling the Physical Dimensions of Peptide Nanotubes by Supramolecular Polymer Coassembly.

Molecular self-assembly of peptides into ordered nanotubes is highly important for various technological applications. Very short peptide building blocks, as short as dipeptides, can form assemblies with unique mechanical, optical, piezoelectric, and semiconductive properties. Yet, the control over nanotube length in solution has remained challenging, due to the inherent sequential self-assembly mechanism. Here, in line with polymer chemistry paradigms, we applied a supramolecular polymer coassembly methodology to modulate peptide nanotube elongation. Utilizing this approach, we achieved a narrow, controllable nanotube length distribution by adjusting the molecular ratio of the diphenylalanine assembly unit and its end-capped analogue. Kinetic analysis suggested a slower coassembly organization process as compared to the self-assembly dynamics of each of the building blocks separately. This is consistent with a hierarchal arrangement of the peptide moieties within the coassemblies. Mass spectrometry analysis demonstrated the bimolecular composition of the coassembled nanostructures. Moreover, the peptide nanotubes' length distribution, as determined by electron microscopy, was shown to fit a fragmentation kinetics model. Our results reveal a simple and efficient mechanism for the control of nanotube sizes through the coassembly of peptide entities at various ratios, allowing for the desired end-product formation. This dynamic size control offers tools for molecular engineering at the nanoscale exploiting the advantages of molecular coassembly.

α-Aminoisobutyric acid incorporation induces cell permeability and antiviral activity of HIV-1 major homology region fragments.

The design of a cell penetrating antiviral peptide, which is derived from the major homology region of HIV-1 capsid protein and includes the non-coded α-aminoisobutyric acid, provides functional evidence for the role of the conserved region in the HIV assembly process and demonstrates the correlation between conformational stability and cellular permeability.