Our SAXS measurements revealed that the spidroin regulation and consequent variability in silk proline corresponded with variations in long period (L) in A. keyserlingi, N. plumipes and P. graeffei, suggesting that spidroin expression and/or proline composition affects nanostructural alignment in the amorphous and lamellar regions. These compositional and structural variations correlated well with extensibility and toughness variations in the silks, providing first evidence of a functional link between spidroin expression, nanostructural formation, and mechanical property variations in the MA silks of different spiders.