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.
Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.
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