Figure 1.
Molecular organization of MiSp.
The amino acid (aa) number of each domain is indicated above the corresponding bar, if it is available. The number of aa in LK domain (n) varies from 83–174. The total number of RP and LK in the MiSp sequence is still unknown and is denoted as “m”. The sequences of the NTD and its adjacent LK domain are unknown.
Figure 2.
Structure and surface plots of CTD.
a) Cartoon drawing of the lowest energy conformer of CTD. The two monomers are indicated by two different colors. b). Hydrophobic and charged surface of CTD. Hydrophobic residues are colored by a scale based on normalized hydrophobicity values [34]: Phe (1.0) for yellow, Val (0.57) for light yellow and Gly (0.0) for white. Positively charged, negatively charged and polar residues are colored by blue, red and light blue.
Table 1.
Experimental restraints and structural statistics for ten lowest-energy NMR structures out of 100 calculated structures.
Figure 3.
Predicted disordered residues (a) and aggregation regions (b) in LKMi.
Disordered residues have scores >0.5. The aggregation-prone regions with aggregation propensity scores >1 are indicated by bars in panel b.
Figure 4.
NMR spectra of LK-CTDMi, CTDMi, RPMi and RP-LK-CTDMi.
a). Overlay of 2D 1H-15N HSQC spectra of isolated CTDMi (red) and di-domain LK-CTDMi (cyan). The signals from the His-tag in the isolated CTDMi were labeled. Correlation peaks from Ala and Gly of the LK domain in LK-CTDMi are clustered in small regions and are indicated by arrows. b). stacked plot of 1D 1H spectra (−0.5 ppm –1.6 ppm) of CTDMi (green, 0.6 mM), RPMi (red, 0.6 mM), RP-LK-CTDMi (cyan, 0.6 mM) and RP-LK-CTDMi (pink, 3 mM).
Figure 5.
Circular Dichroism (CD) spectrum of RPMi.
Figure 6.
Urea-induced denaturation of MiSp fragments under different NaCl concentrations monitored by CD.
a) CTDMi; b) LK-CTDMi; c) RPMi; d) RP-LK-CTDMi. The lines represent the fitting curves. All the data were normalized.
Figure 7.
Urea-induced denaturation of CTDMi mutants. The solid lines represent the fitting curves.
Figure 8.
Shear-force-induced aggregation of MiSp fragments and MBP monitored by OD350 (a), dependence of aggregation of RP-LK-CTDMi on salts (b), and SEM of silk fibers formed by RP-LK-CTDMi (c).
The scale bar is 10 µm.