The Structural Characterization Of Argiope Trifasciata Spider Wrapping Silk By Solution-State NMR
Biomolecular nuclear magnetic resonance (NMR) spectroscopy frequently employs secondary chemical shifts to estimate local secondary structuring in advance of obtaining a full 3D structure. To assess the effect of variation in dielectric upon secondary chemical shift, two new sets of random coil chemical shifts, one in dimethyl sulfoxide and the other in chloroform:methanol:water, a popular membrane-mimetic solvent system, were produced. Using a new program, CS-CHEMeleon, we demonstrated that the chemical shift-based structure prediction accuracy was much more affected by the secondary structure type than solvent environment. Spider silks are outstanding biomaterials with remarkable mechanical and physical properties. Little is known about spider silk atomic-level structuring and how this relates to variations in mechanical properties. The spider wrapping silk protein in Argiope trafisciata (AcSp1) contains a 200 amino acid sequence (“W” unit) repeated at least 14 times; it self-assembles into the toughest type of silk. The high-resolution structure of a recombinantly produced repeat W unit was solved by solution-state NMR. W1 is composed of a 5-helix globular core with intrinsically disordered N- and C-terminal tails. With the use of split-intein technology, the effect of repetitive domain tandemization was investigated with W2 (W1+W1) through separate selective labeling of each of the two repeats in W2 with 13C and/or 15N NMR active isotopes, providing W2 constructs where only one W unit was enriched with NMR-active isotopes. W2 backbone chemical shifts demonstrate the conservation of the W1 structure within W2 and nuclear spin relaxation analysis demonstrates that motions within W2 are conserved with tandemerization. Reduced spectral density mapping analysis shows two types of motion, one for globular core and another for the tails or linker, further emphasizing the two separate domains of AcSp1. NMR titrations of W1 and W2 with chemical denaturants and with the detergent dodecylphosphocholine (DPC) were performed to locate the residues most amenable to unfolding and, hence, likely responsible for initial structural transition upon fibrillogenesis. On the basis of these data, a structural model for the solution structure of native AcSp1 is described, having a “beads-on-a-string” architecture, and the region likely responsible for seeding fibrillogenesis is proposed.