Production and Properties of Triple Chimeric Spidroins

All spider silk proteins (spidroins) are composed of N- and C-terminal domains (NT and CT) that act as regulators of silk solubility and assembly and a central repetitive region, which confers mechanical properties to the fiber. Among the seven types of spider silks, aciniform silk has the highest toughness. Herein, we fused NT and CT domains from major and minor ampullate spidroins (MaSps and MiSps), respectively, to 1–4 repeat domains (W) from another type of spidroin, aciniform spidroin 1­(AcSp1). Although the three domains originate from distantly related spidroin types, they keep their respective characteristics in the chimeric spidroins. Furthermore, all chimeric spidroins could form silk-like fibers by manual-drawing. In contrast to fibers made in the same manner from W domains only, NTW_1–4CT fibers show superior mechanical properties. Our results suggest that chimeric spidroins with NT, CT, and repeat domains can be designed to form fibers with various mechanical properties

Protective Effects of Dimethyl Sulfoxide on Labile Protein Interactions during Electrospray Ionization

Electrospray ionization mass spectrometry is a valuable tool to probe noncovalent interactions. However, the integrity of the interactions in the gas-phase is heavily influenced by the ionization process. Investigating oligomerization and ligand binding of transthyretin (TTR) and the chaperone domain from prosurfactant protein C, we found that dimethyl sulfoxide (DMSO) can improve the stability of the noncovalent interactions during the electrospray process, both regarding ligand binding and the protein quaternary structure. Low amounts of DMSO can reduce in-source dissociation of native protein oligomers and their interactions with hydrophobic ligands, even under destabilizing conditions. We interpret the effects of DMSO as being derived from its enrichment in the electrospray droplets during evaporation. Protection of labile interactions can arise from the decrease in ion charges to reduce the contributions from Coulomb repulsions, as well as from the cooling effect of adduct dissociation. The protective effects of DMSO on labile protein interactions are an important property given its widespread use in protein analysis by electrospray ionization mass spectrometry (ESI-MS)

Contact map of the Aβ-Dec-DETA complex.

<p>The probability (0.0≤<i>P</i><0.6) of the contact between the center of geometry of sidechain heavy atoms of each Aβ residue and each Dec-DETA heavy atom is colored (white to blue grids). The probability was calculated using the data obtained from the whole simulations of all ten trajectories. The Aβ residues and Dec-DETA atoms corresponding to the X and Y-axis numbers, respectively, are listed below the map.</p

Fractions of polar and nonpolar contacts between Aβ and Dec-DETA or Pep1b for each peptide-conformation class^a.

<p>aThe fractions were calculated for the three peptide-conformation classes ((1) RMSD<2.0 Å, (2) 2.0 Å≤RMSD<4.0 Å, and (3) RMSD≥4.0 Å) using all ten trajectories of each system. The occurrence of the polar or nonpolar contacts for each peptide-conformation class was divided by the frequency of each peptide-conformation class.</p><p>bThe polar contacts were determined by the existence of at least one HB between Aβ and Dec-DETA or Pep1b.</p><p>cThe nonpolar contacts were determined by the existence of at least one C-C or C-N contact between the Aβ middle nonpolar part and the nonpolar part of Dec-DETA or Pep1b.</p

Average number of polar and nonpolar contacts between Aβ and Dec-DETA or Pep1b for each peptide-conformation class^a.

<p>aThe mean values (± standard deviations) were calculated for the three peptide-conformation classes ((1) RMSD<2.0 Å, (2) 2.0 Å≤RMSD<4.0 Å, and (3) RMSD≥4.0 Å) using all ten trajectories of each system.</p><p>bAveraged over periods with at least one HB between Aβ and Dec-DETA or Pep1b.</p><p>cAveraged over periods with at least one C-C or C-N contact between the Aβ middle nonpolar part and the nonpolar part of Dec-DETA or Pep1b.</p

Structural changes of trajectory 4 of the Aβ-Dec-DETA system.

<p>The RMSD and R_g of the Aβ middle region (A) and the number of HBs between Aβ and Dec-DETA (B) are shown. The structure obtained at 14.29 ns (with large RMSD (4.59 Å), large R_g (8.48 Å), and four HBs) is also shown (C).</p

Structural changes of trajectory 5 of the Aβ-Pep1b system.

<p>The RMSD and R_g of the Aβ middle region (A) and the number of HBs between Aβ and Pep1b (B) are shown. The structures obtained at 5.15 ns (with large RMSD (4.10 Å), large R_g (7.56 Å), and seven HBs), at 6.36 ns (with medium RMSD (3.64 Å), small R_g (6.16 Å), and six HBs), at 9.12 ns (with large RMSD (4.36 Å), large R_g (7.97 Å), and two HBs), and at 10.47 ns (with medium RMSD (3.45 Å), small R_g (6.56 Å), and four HBs) are also shown (C).</p

Initial structures of Aβ and the Aβ-ligand complexes.

<p>The initial energy-minimized structures of Aβ (A), the Aβ-Dec-DETA complex (B), and the Aβ-Pep1b complex (C) are shown. The positions of Aβ backbones (ribbons), Aβ sidechains (lines), and the ligands (lines and balls) are displayed. The structural formulae of Dec-DETA (B) and Pep1b (C) are also shown. The numbering of carbon (gray), nitrogen (blue), and oxygen (red) atoms is indicated. The residues of Aβ with which the different groups of the ligands are designed to interact (arrows) are also indicated.</p

Timelines of Aβ-ligand contacts.

<p>Timelines showing the presence of at least one Aβ-ligand hydrogen bonding contact for Aβ-Dec-DETA (A) and Aβ-Pep1b (B), and for Aβ-Pep1b (C) also when both kinds of HBs between Aβ and Pep1b (between the Aβ acidic residue sidechains and the Pep1b basic functional groups, and between the Aβ basic residue sidechains and the Pep1b acidic functional groups) were formed at the same time. The ligand-binding events are distinguished by using different colors for the three peptide-conformation classes 1 (black bars), 2 (gray bars), and 3 (red bars).</p

Histograms of RMSD of Aβ in the absence or presence of the ligands.

<p>The histograms of the Aβ (black bars), Aβ-Dec-DETA (blue bars), and Aβ-Pep1b (green bars) systems are shown. The histograms were obtained using the data of the whole simulations (A) and the second half of the simulations (B) of all ten trajectories of each system. The relative frequencies of the appearance of the Aβ structures sorted out by the three levels of RMSD (RMSD<2.0 Å, 2.0 Å≤RMSD<4.0 Å, and RMSD≥4.0 Å) of the Aβ middle region are indicated. The relative frequencies were calculated against total time of all ten trajectories of each system.</p