Tabot M. D. Besong

Protein-like oligomerization of carbohydrates.

Sugar can, too: Many proteins form noncovalent and thermodynamically reversible oligomers, which can dictate a proteins functionality. For the first time, the presence of multiple oligomeric forms is shown in a whole class of polymeric carbohydrates, the 6-deoxy-6-aminocelluloses, using analytical ultracentrifugation as a probe.

Assembly of the Yeast Exoribonuclease Rrp6 with Its Associated Cofactor Rrp47 Occurs in the Nucleus and Is Critical for the Controlled Expression of Rrp47

Background: The Rrp6·Rrp47 complex is important for RNA processing and degradation events. Results: Rrp6 and Rrp47 are independently imported into the nucleus, and Rrp47 is destabilized in cells lacking Rrp6. Conclusion: Rrp6 binds Rrp47 in the nucleus and protects it from proteolysis. Significance: Nuclear assembly of the Rrp6·Rrp47 complex spatially limits the nuclease complex and controls the expression of Rrp47. Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6·Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.

Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction

Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1N), α-5 (hLM α-5N) and β-3 (hLM β-3N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.

Evidence for self-association of a miniaturized version of agrin from hydrodynamic and small-angle X-ray scattering measurements.

Hydrodynamic studies of miniagrin indicate a molar mass that is 20% larger than the value calculated from the sequence of this genetically engineered protein. Consistent with this finding is the negative sign and also the magnitude of the second virial coefficient obtained from small-angle X-ray scattering measurements. The inference that miniagrin reversibly self-associates is confirmed by a sedimentation equilibrium study that yields an equilibrium constant of 0.24 L/g for a putative monomer-dimer interaction. Finally, Guinier analysis of the small-angle X-ray scattering (SAXS) results yields concentration-dependent values for the radius of gyration that may be described by the monomer-dimer model and respective R(g) values of 40 and 105 Å for the monomeric and dimeric miniagrin species. Although intermolecular protein interactions are endemic in the events leading to acetylcholine receptor aggregation by agrin, the matrix proteoglycan of which miniagrin is a miniaturized model, this investigation raises the possibility that agrin may itself self-associate.

The effective time of centrifugation for the analysis of boundary spreading in sedimentation velocity experiments

This investigation establishes a likely order of magnitude for the zero-time correction factor governing the effective time of centrifugation that is pertinent in the analysis of boundary spreading in sedimentation velocity experiments. This correction is shown to be too small to unduly affect the magnitudes of sedimentation and diffusion coefficients deduced from the application of computer software incorporating the printout value of ω²t and an effective position of the air-solution meniscus that is obtained as an additional parameter in the analysis involving nonlinear least-squares curve-fitting of sedimentation velocity distributions to the Lamm equation. Although this procedure slightly underestimates the actual meniscus position (r(a)), uncertainty about its exact location precludes adoption of the alternative approach with r(a) fixed and the correction to ω²t regarded as the additional curve-fitting parameter.

Matrix-free hydrodynamic study on the size distribution and conformation of three technical lignins from wood and non-wood

Abstract Molecular weight (MW) and related conformational data of three commercially available technical lignins (Alcell L, kraft L, and soda L) have been studied by means of analytical ultracentrifugation, taking advantage of some recent developments in both sedimentation velocity and sedimentation equilibrium determinations. The lignins were dissolved in dimethyl sulphoxide (with ca. 90% solubility), and solutions were studied with regards to their oligomeric state, heterogeneity profiles (distribution of sedimentation coefficients), and molecular weight distributions (MWD). Alcell L and soda L have similar properties showing one major low MW component and two minor high MW components, whereas kraft L appears to be larger and more uniform, i.e., it shows a more monodisperse MWD. Weight average molecular weight (Mw) data from sedimentation equilibrium obtained by the new SEDFIT-MSTAR procedure in conjunction with MULTISIG analysis were found to be ~18 kDa (Alcell L), 25 kDa (kraft L), and 15 kDa (soda L). Further analysis of the data by means of the routines MULTISIG and M_INVEQ confirmed the presence of additional components in Alcell L and soda L, and the larger size and high degree of monodispersity of kraft L. The intrinsic viscosity data of the three lignins were found to be very similar in the range of 22–24 ml g-1, and all data were consistent with an elongated plate shape molecular structure with an equivalent discoid aspect ratio ~30.

Corrigendum: Genetic improvement of tomato by targeted control of fruit softening

Nat. Biotechnol. 34, 950–952 (2016); published online 25 July 2016; corrected after print 14 September 2016 In the version of this article initially published, the volume and page numbers for reference 46 were incorrect. The error has been corrected in the HTML and PDF versions of the article

The effect of thermal processing in oil on the macromolecular integrity and acrylamide formation from starch of three potato cultivars organically fertilized

Abstract Starches from three organically produced cultivars of potato tuber (Lady Rosetta, Spunta and Voyager) have been studied in relation to (i) acrylamide production (ii) macromolecular integrity after frying with extra virgin olive oil, soybean oil and corn oil. During cultivation, a treatment involving the combination of nitrogen, phosphorus and potassium fertilization under organic farming was applied (N1, P2, K1 where Ν1 = 1.3 g Ν per plant, P2 = 5.2 g P2O5 per plant, Κ1 = 4.0 g K2O per plant). Potatoes fried in olive oil retained the highest glucose concentrations for all cultivars 0.85 ± 0.2 mmol/kg, followed by 0.48 ± 0.2 for those fried in corn oil and 0.40 ± 0.1 mmol/kg for those fried in soybean oil. The highest average fructose concentration was recorded for the samples fried in corn oil as 0.81 ± 0.2, followed by 0.80 ± 0.2 and 0.68 ± 0.3 mmol/kg for the samples fried in olive and soybean oils, respectively. Asparagine was the most abundant free amino acid in the three varieties tested, followed by glutamine and aspartic acid. The mean initial concentration of asparagine in raw potatoes tubers was 42.8 ± 1.6 mmoles kg−1 for Lady Rosetta, 34.6 ± 1.2 mmoles kg−1 (dry weight) for Spunta and 36.2 ± 2.0 mmoles kg−1 for Voyager. Lady Rosetta contained a significantly higher concentration of asparagine compared to the other two varieties (p < 0.05). The greatest quantity of acrylamide was observed in French fries derived from the potato variety Lady Rosetta when fried in soybean oil and it was 2,600 ± 440 μg/kg, followed by Spunta which was 2,280 ± 340 μg/kg and Voyager 1,120 ± 220 μg/kg. There is a significant reduction in the formation of acrylamide in the variety Voyager compared to the others (p = 0.05).