Fig. 2: Biophysical analysis of human mini-collagen VI α1α2α3C1C2.
a Schematic representation of the mini-collagen VI α1α2α3C1C2 constructs. These included N-terminal 6x His, Twin-Strep or FLAG-tags in the α1, α2 and α3 chains, respectively, followed by 9x Gly-X-Y triplets and the C1 and C2 vWA domains, with the furin cleavage site between the α3 chain C1–C2 domains mutated to prevent cleavage. b Coomassie-stained SDS-PAGE gel of the purified α1α2α3C1C2 heterotrimer under reduced (R) and non-reduced (NR) conditions. The trimer dissociates into the three individual α-chains upon reduction of intermolecular disulphide bonds, as shown by three bands of the same intensity. Purifications were independently repeated at least 3 times with similar results. c SEC-Multi-angle light scattering of the purified α1α2α3C1C2 heterotrimer indicated a molecular weight of 155.5 kDa, comparable to its predicted molecular weight of 158.9 kDa. d Mass photometry also confirmed a single homogeneous species. e AlphaFold prediction of the α1α2α3C1C2 heterotrimer is shown in cartoon representation in two orthogonal orientations. The α1, α2 and α3 chains are coloured green, cyan and magenta, respectively. f Experimental X-ray scattering data of the α1α2α3C1C2 heterotrimer (Iexp, grey) plotted as a function of q, compared to the theoretical scattering of the AlphaFold model shown in black (Imodel) with χ2 = 1.76. g The low q scattering data of the α1α2α3C1C2 heterotrimer is represented as a Guinier plot showing the linearity of the data across the Guinier region, as defined by dashed lines (qRg < 0.65 and qRg ≈ 1.3). h Pair-distance distribution function P(r) for the α1α2α3C1C2 heterotrimer with maximum dimension (Dmax) of 145 Å. Source data are provided as a Source data file.
