Structural Basis of Oligomerization of N-Terminal Domain of Spider Aciniform Silk Protein.

Spider silk is self-assembled from water-soluble silk proteins through changes in the environment, including pH, salt concentrations, and shear force. The N-terminal domains of major and minor ampullate silk proteins have been found to play an important role in the assembly process through salt- and pH-dependent dimerization. Here, we identified the sequences of the N-terminal domains of aciniform silk protein (AcSpN) and major ampullate silk protein (MaSpN) from Nephila antipodiana (NA). Different from MaSpN, our biophysical characterization indicated that AcSpN assembles to form large oligomers, instead of a dimer, upon condition changes from neutral to acidic pH and/or from a high to low salt concentration. Our structural studies, by nuclear magnetic resonance spectroscopy and homology modelling, revealed that AcSpN and MaSpN monomers adopt similar overall structures, but have very different charge distributions contributing to the differential self-association features. The intermolecular interaction interfaces for AcSp oligomers were identified using hydrogen-deuterium exchange mass spectrometry and mutagenesis. On the basis of the monomeric structure and identified interfaces, the oligomeric structures of AcSpN were modelled. The structural information obtained will facilitate an understanding of silk fiber formation mechanisms for aciniform silk protein.

Atomistic Insights into the Functional Instability of the Second Helix of Fatty Acid Binding Protein.

Structural dynamics of fatty acid binding proteins (FABPs), which accommodate poorly soluble ligands in the internalized binding cavities, are intimately related to their function. Recently, local unfolding of the α-helical cap in a variant of human intestinal FABP (IFABP) has been shown to correlate with the kinetics of ligand association, shedding light on the nature of the critical conformational reorganization. Yet, the physical origin and mechanism of the functionally relevant transient unfolding remain elusive. Here, we investigate the intrinsic structural instability of the second helix (αII) of IFABP in comparison with other segments of the protein using hydrogen-exchange NMR spectroscopy, microsecond molecular dynamics simulations, and enhanced sampling techniques. Although tertiary interactions positively contribute to the stability of helices in IFABP, the intrinsic unfolding tendency of αII is encoded in its primary sequence and can be described by the Lifson-Roig theory in the absence of tertiary interactions. The unfolding pathway of αII in intact proteins involves an on-pathway intermediate state that is characterized with the fraying of the last helical turn, captured by independent enhanced sampling methods. The simulations in this work, combined with hydrogen-exchange NMR data, provide new, to our knowledge, atomistic insights into the functional local unfolding of FABPs.