This study investigates the structural conversion of the C-terminal (CT) domain of the major ampullate spidroin 1 (MaSp1) during the assembly of spider silk fibers. The CT domain, a non-repetitive and highly conserved segment of MaSp1, is crucial for initiating the self-assembly of spidroins into fibers. Structural investigations using recombinant CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation, with helix N°4 being the most likely segment to initiate this structural conversion. This prediction is supported by the ability of a peptide corresponding to helix N°4 to undergo pH-induced conversion into β-sheets and self-assemble into nanofibrils. The CT domain can self-assemble into fibers under similar conditions to the recombinant 4RepCT protein, although with lower stability. The CT domain is composed of approximately 100 amino acids and contains a highly conserved hydrophobic region, which may be essential for protein folding and fiber formation. Structural analyses using solid-state methods, including FTIR, X-ray diffraction, and NMR, reveal that the CT domain in fiber form contains a mixture of α-helices and β-sheets, with a significant β-sheet content. The X-ray diffraction pattern of CT fibers shows a distinct nanocrystalline component, with inter-sheet distances wider than those observed in β-poly(L-Ala) crystalline patterns. The CT domain fibers bind a molecular probe for amyloid detection, suggesting the presence of ordered β-sheet structures similar to amyloid-like fibrils. Computational analysis using ZipperDB and TANGO algorithms indicates that helix N°4 has a higher propensity to form β-sheet structures than the rest of the sequence. A synthetic peptide corresponding to helix N°4 shows pH-dependent α-to-β structural conversion and can assemble into nanofibrils, contributing to the fundamental architecture of spider silk. The study highlights the importance of the CT domain in the structural transition from α-helix to β-sheet during fiber assembly. The findings suggest that the CT domain plays a key role in triggering the structural conversion of spidroins during fiber formation, with helix N°4 being a critical segment for this process. The results provide insights into the structural organization of spider silk and have implications for the development of biomimetic materials.This study investigates the structural conversion of the C-terminal (CT) domain of the major ampullate spidroin 1 (MaSp1) during the assembly of spider silk fibers. The CT domain, a non-repetitive and highly conserved segment of MaSp1, is crucial for initiating the self-assembly of spidroins into fibers. Structural investigations using recombinant CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation, with helix N°4 being the most likely segment to initiate this structural conversion. This prediction is supported by the ability of a peptide corresponding to helix N°4 to undergo pH-induced conversion into β-sheets and self-assemble into nanofibrils. The CT domain can self-assemble into fibers under similar conditions to the recombinant 4RepCT protein, although with lower stability. The CT domain is composed of approximately 100 amino acids and contains a highly conserved hydrophobic region, which may be essential for protein folding and fiber formation. Structural analyses using solid-state methods, including FTIR, X-ray diffraction, and NMR, reveal that the CT domain in fiber form contains a mixture of α-helices and β-sheets, with a significant β-sheet content. The X-ray diffraction pattern of CT fibers shows a distinct nanocrystalline component, with inter-sheet distances wider than those observed in β-poly(L-Ala) crystalline patterns. The CT domain fibers bind a molecular probe for amyloid detection, suggesting the presence of ordered β-sheet structures similar to amyloid-like fibrils. Computational analysis using ZipperDB and TANGO algorithms indicates that helix N°4 has a higher propensity to form β-sheet structures than the rest of the sequence. A synthetic peptide corresponding to helix N°4 shows pH-dependent α-to-β structural conversion and can assemble into nanofibrils, contributing to the fundamental architecture of spider silk. The study highlights the importance of the CT domain in the structural transition from α-helix to β-sheet during fiber assembly. The findings suggest that the CT domain plays a key role in triggering the structural conversion of spidroins during fiber formation, with helix N°4 being a critical segment for this process. The results provide insights into the structural organization of spider silk and have implications for the development of biomimetic materials.