Westerhausen A, Kishi J, Prockop D J
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1990 Aug 15;265(23):13995-4000.
Cultured skin fibroblasts from two probands with lethal variants of osteogenesis imperfecta synthesized type I procollagen that was posttranslationally over-modified. Analysis of cDNAs and genomic DNAs from the two probands demonstrated that proband I had a single-base mutation that converted the codon for glycine alpha 1-631 to a codon for serine, and proband II had a single-base mutation that converted the codon for glycine alpha 1-598 to a codon for serine. Although the two serine-for-glycine substitutions were separated by only 35 residues, they had markedly different effects on the thermal unfolding of the collagen triple helix as assayed by brief protease digestion. The type I procollagen from proband I (serine alpha 1-631) had an essentially normal temperature for thermal unfolding. In contrast, type I procollagen from proband II (serine alpha 1-598) was cleaved to readily identifiable intermediate fragments of about 630 residues at 20 degrees C. With procollagens from both probands, collagenase A fragments containing the first 775 amino acids of the alpha chain domains had a lowered temperature for thermal unfolding as assayed by brief protease digestion. The collagenase A fragments from proband I were cleaved to intermediates of about 600 amino acids at 36 degrees C and to fragments of about 510 residues at 37 degrees C. The collagenase A fragments from proband II were cleaved to intermediates of about 630 residues at 32 degrees C, to fragments of about 600 residues at 36 degrees C, and to fragments of about 510 at 37 degrees C. The fragments of about 510 residues from both mutated procollagens were more stable to protease digestion than the collagenase A fragments of 775 residues from normal type I collagen. The results demonstrate that the effects of glycine substitutions on the thermal unfolding of type I collagen are highly position-specific. They also provide direct evidence for previous indications that the triple helix of the protein undergoes micro-unfolding of a series of relatively independent "cooperative blocks" in the predenaturation range of temperatures.
来自两名患有致死性成骨不全变异体的先证者的培养皮肤成纤维细胞合成了翻译后过度修饰的I型前胶原。对两名先证者的cDNA和基因组DNA分析表明，先证者I有一个单碱基突变，将甘氨酸α1-631的密码子转换为丝氨酸密码子，先证者II有一个单碱基突变，将甘氨酸α1-598的密码子转换为丝氨酸密码子。尽管这两个丝氨酸取代甘氨酸的位点仅相隔35个残基，但通过短暂蛋白酶消化测定，它们对胶原三螺旋的热解折叠有明显不同的影响。先证者I的I型前胶原（丝氨酸α1-631）热解折叠温度基本正常。相比之下，先证者II的I型前胶原（丝氨酸α1-598）在20℃时被切割成易于识别的约630个残基的中间片段。对于两名先证者的前胶原，通过短暂蛋白酶消化测定，含有α链结构域前775个氨基酸的胶原酶A片段热解折叠温度降低。先证者I的胶原酶A片段在36℃时被切割成约600个氨基酸的中间片段，在37℃时被切割成约510个残基的片段。先证者II的胶原酶A片段在32℃时被切割成约630个残基的中间片段，在36℃时被切割成约600个残基的片段，在37℃时被切割成约510个残基的片段。两种突变前胶原约510个残基的片段比正常I型胶原775个残基的胶原酶A片段对蛋白酶消化更稳定。结果表明，甘氨酸取代对I型胶原热解折叠的影响具有高度的位置特异性。它们还为先前的迹象提供了直接证据，即该蛋白质的三螺旋在变性前温度范围内经历一系列相对独立的“协同结构域”的微解折叠。
