Pulling geometry defines the mechanical resistance of a beta-sheet protein

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.     

Physiological responses of carbon-sequestering microalgae to elevated carbon regimes

In order to identify a high carbon-sequestering microalgal strain, the physiological effect of different concentrations of carbon sources on microalgae growth was investigated. Five indigenous strains (I-1, I-2, I-3, I-4 and I-5) and a reference strain (I-0: Coccolithus pelagicus 913/3) were subjected to CO₂ concentrations of 0.03–15% and NaHCO₃ of 0.05–2 g CO₂ l^–1. The logistic model was applied for data fitting, as well as for estimation of the maximum growth rate (μ_max) and the biomass carrying capacity (B_max). Amongst the five indigenous strains, I-3 was similar to the reference strain with regards to biomass production values. The B_max of I-3 significantly increased from 214 to 828 mg l^–1 when CO₂ concentration was increased from 0.03 to 15% (r = 0.955, P = 0.012). Additionally, the B_max of I-3 increased with increasing NaHCO₃ (r = 0.885, P = 0.046) and was recorded at 153 mg l^–1 (at 0.05 g CO₂ l^–1) and 774 mg l^–1 at (2 g CO₂ l^–1). Relative electron transport rate (rETR) and maximum quantum yield (F_v/F_m) were also applied to assess the impact of elevated carbon sources on the microalgal cells at the physiological level. Isolate I-3 displayed the highest rETR confirming its tolerance to higher quantities of carbon. Additionally, the decline in F_v/F_m with increasing carbon was similar for strains I-3 and the reference strain. Based on partial 28s ribosomal RNA gene sequencing, strain I-3 was homologous to the ribosomal genes of Chlorella sp.     

Air travel and venous thrombosis: how much help might aspirin be?

There has been considerable attention focused recently on the risk of deep venous thrombosis (DVT) associated with air travel. Despite the lack of evidence among air travelers, a single dose of aspirin has been widely recommended as a means of preventing such thrombosis. We have calculated the potential benefit of aspirin by applying the data for aspirin in preventing DVT in hip fracture patients to the estimated rates of travel-related DVT. If the rate of travel-related DVT is 20 per 100,000 travelers, then we will have to treat 17,000 people with aspirin to prevent 1 additional DVT.     

COPT6 Is a Plasma Membrane Transporter That Functions in Copper Homeostasis in Arabidopsis and Is a Novel Target of SQUAMOSA Promoter-binding Protein-like 7

Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma membrane and mediates copper accumulation when expressed in the Saccharomyces cerevisiae copper uptake mutant. Although the primary sequence of COPT6 contains the family conserved domains, including methionine-rich motifs in the extracellular N-terminal domain and a second transmembrane helix (TM2), it is different from the founding family member, S. cerevisiae Ctr1p. This conclusion was based on the finding that although the positionally conserved Met¹⁰⁶ residue in the TM2 of COPT6 is functionally essential, the conserved Met²⁷ in the N-terminal domain is not. Structure-function studies revealed that the N-terminal domain is dispensable for COPT6 function in copper-replete conditions but is important under copper-limiting conditions. In addition, COPT6 interacts with itself and with its homolog, COPT1, unlike Ctr1p, which interacts only with itself. Analyses of the expression pattern showed that although COPT6 is expressed in different cell types of different plant organs, the bulk of its expression is located in the vasculature. We also show that COPT6 expression is regulated by copper availability that, in part, is controlled by a master regulator of copper homeostasis, SPL7. Finally, studies using the A. thaliana copt6-1 mutant and plants overexpressing COPT6 revealed its essential role during copper limitation and excess.