Location of Glycine Mutations within a Bacterial Collagen Protein Affects Degree of Disruption of Triple-helix Folding and Conformation

Abstract

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa) n sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2°C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triplehelix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

Inherited Connective Tissue Disorders of Collagens: Lessons from Targeted Mutagenesis

“…However, the glycine substitution G1519D located in another segment of the triple helix had no effect on procollagen VII secretion or its ability to anchor fibril assembly. These data showed that the biological impact of glycine substitutions can depend on their position within the triple helix, as shown for collagen I (Cheng et al, 2011).…”

“…A very recent study utilized a recombinant bacterial collagen to develop a mutagenesis scheme in which a glycine residue within the triple-helix sequence is substituted with arginine or serine. The purpose was to analyze the positional effect of glycine mutations on triple-helix formation and stability (Cheng et al, 2011). Interestingly, all glycine mutations provoked a significant delay in the triple-helix formation.…”

Functionalised type-I collagen as a hydrogel building block for bio-orthogonal tissue engineering applications

“…The measured CD spectrum of MAC resulted in similar spectra to that of native collagen spectra with an Rpn value of 0.13 indicating the retention of the triple helical assembly after >85% modification of collagen's lysines. 32,33 Sameness in Rpn values after methacrylate modification indicates the specific functionalization of collagen at ε amines of lysine that did not alter the triple helical propensity. From our CD spectroscopic analysis it is evident that the intrinsic structure of collagen was largely retained after modification (Fig.…”

Bacterial collagen-like proteins that form triple-helical structures

“…The recombinant bacterial collagen system has been applied to characterize the effects of a Gly mutation, since a mutation can be introduced at any location within the triple-helix while controlling the sequence surrounding it (Cheng et al 2011). Site-directed mutagenesis was used to introduce a Gly→Arg or a Gly→Ser mutation at a site near the middle or near the N-terminus of the triple-helix adjacent to the trimerization domain.…”