Spider silk’s combination of strength and biocompatibility puts chemists’ best polymers to shame. But it’s still not clear how arachnids polymerize soluble protein monomers called spidroins into silk fibers. Hannes Neuweiler of Julius Maximilians University, in Würzburg, Germany, and colleagues zeroed in on one aspect of that question: the N-terminal domain of spidroin 1, a protein region many different spider species have in common (J. Am. Chem. Soc. 2014, DOI: 10.1021/ja508760a). This region contains a structural switch that facilitates polymerization in response to a pH change in a spider’s spinning duct. Multiple labs have studied the region with crystallography, but Neuweiler’s team wanted a technique that could provide more insight into the protein’s motions, which are too tiny for the resolution limit of single-molecule fluorescence spectroscopy. The researchers engineered tryptophan-fluorophore pairs that could follow 1-nm-scale motions in the domain and combined those probes with correlation analysis of single-molecule fluorescence fluctuations. In tests on isolated proteins, the team learned that conformational changes in the N-terminal domain occur while the protein is still in monomer form, in contrast to some prior studies that suggest association during polymerization kicks off conformation changes.