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Collagen IV, also called type IV collagen, is the network-forming collagen that provides a major scaffold of basement membranes. Three polypeptide chains assemble into a molecular unit, and units associate through their ends into a sheet-like network. This organization suits a thin supporting layer beneath an epithelium, rather than a long cable made of parallel molecules.^[ref]

The Collagen IV NC1 domain is a noncollagenous end region where adjoining molecular units meet. A sulfilimine bond links a methionine side chain in one unit to a hydroxylysine side chain in another. Thus the scaffold combines molecular association with a chemically specific covalent bond.^[ref]

One can distinguish two questions about a basement membrane: which proteins form its scaffold, and how neighboring units are fastened together. Isolating linked peptides and comparing their mass spectrometry and nuclear magnetic resonance signals answered the second question. The investigation connected a tissue-scale network to the identities of individual amino acid residues.^[ref]

The Collagen IV NC1 domain is the noncollagenous protein domain at the C-terminus of a Collagen IV chain. NC1 regions participate in the end-to-end assembly of the network that supports a basement membrane. A three-chain Collagen IV protomer presents three NC1 regions at this end; association of two such ends forms a six-subunit Collagen IV NC1 hexamer.^[ref]

The counting is useful: a six-subunit junction joins two three-chain molecular units, rather than requiring each Collagen IV molecule to contain six chains. The Collagen IV NC1 hexamer describes a local meeting point in the larger scaffold.

The junction is reinforced by sulfilimine bonds between methionine and hydroxylysine residues of adjoining units. In 2009, mass spectrometry and nuclear magnetic resonance measurements on isolated linked peptides identified this bond. Chemical reduction separated the linked peptides, providing another test of the proposed structure. Up to six such links can reinforce the interface between the two NC1 ends.^[ref]

A Collagen IV protomer is a three-chain molecular unit of Collagen IV that assembles with other units to form a basement membrane network. Much of its length has a collagen triple helix, while its C-terminus presents three Collagen IV NC1 domains. Association of two such ends forms a Collagen IV NC1 hexamer.^[ref]

The organizational levels can be counted separately. Ten intact three-chain Collagen IV protomers contain thirty polypeptide chains, but this count does not describe how the ten units are connected. A six-subunit NC1 junction joins two protomers at one end; it does not convert either protomer into a six-chain molecule.

The term is therefore useful when moving between a protein's molecular composition and the architecture of the scaffold it builds. Identifying a three-chain unit answers a different question from identifying the contacts and covalent bonds between neighbouring units.

Hydroxylysine is a hydroxylated derivative of the amino acid lysine. In the important collagen-associated form, a hydroxyl group is attached at carbon 5 of the side chain. Free 5-hydroxylysine has formula ${\mathrm{C}}_6{\mathrm{H}}_{14}{\mathrm{N}}_2{\mathrm{O}}_3$, and its connectivity can be writtenThis formula specifies the arrangement of the groups but does not by itself distinguish the possible stereoisomers. ^[ref]

In collagen, hydroxylysine occurs as a modified amino acid residue within a protein. A concrete example is the sulfilimine bond linking hydroxylysine-211 of one Collagen IV unit to methionine-93 of another. Mass spectrometry and nuclear magnetic resonance identified that sulfur–nitrogen connection.^[ref] The example shows why a modified side chain matters: changing a particular residue changes the chemistry available for joining protein units. Free hydroxylysine and a residue incorporated into collagen must nevertheless be distinguished, because incorporation places its backbone in peptide bonds.

Roberto Vanacore investigates the chemistry of the extracellular matrix, particularly the collagen networks supporting tissues. A 2009 study led by Vanacore identified an unusual sulfilimine bond joining neighboring Collagen IV units. The team combined mass spectrometry with nuclear magnetic resonance to establish a sulfur–nitrogen connection between methionine and hydroxylysine.^[ref]

The discovery makes a useful distinction between seeing that two proteins are joined and determining the chemical identity of the join. Precise mass constrains the atoms present, while spectroscopic measurements provide additional evidence about their arrangement. Together these methods resolved a link whose identification had been difficult from structural images alone. The crosslink reinforces the Collagen IV scaffold of basement membranes, connecting atomic bonding with tissue-scale mechanical organization.^[ref]