Mohs Angela, Popiel Magdalena, Li Yingjie, Baum Jean, Brodsky Barbara
Department of Biochemistry, University of Medicine and Dentistry of New Jersey (UMDNJ)-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854.
J Biol Chem. 2006 Jun 23;281(25):17197-17202. doi: 10.1074/jbc.M601763200. Epub 2006 Apr 12.
Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.
纤维状胶原蛋白对甘氨酸有绝对需求，即每三个残基中有一个甘氨酸，而非纤维状胶原蛋白（如基底膜中的IV型胶原蛋白）的三螺旋结构域中通常会出现甘氨酸-X-Y重复模式的中断。在本研究中，设计了一个30聚体肽模型，其中包含在IV型胶原蛋白α5链中发现的中断序列GPOGAAVMGPOGPO。GAAVM肽形成稳定的三螺旋结构，其熔解温度（Tm）为29℃。与每三个残基中有一个甘氨酸的对照肽相比，GAAVM肽的圆二色光谱在225nm处的最大吸收显著降低，稳定性下降了10℃。观察到量热焓降低了50%，这可能是由于由规则排列的主链羰基锚定的有序水结构被破坏所致。对GAAVM肽中特定的15N标记残基进行的核磁共振研究表明，中断序列两侧的甘氨酸-脯氨酸-羟脯氨酸残基具有正常的三螺旋结构。中断序列内的序列并非无序，但显示出氢交换速率改变和缬氨酸化学位移异常。此前有报道称，设计用于模拟肽（脯氨酸-羟脯氨酸-甘氨酸）10中的类似中断（GPOGPOPOGPO）的肽无法形成稳定的三螺旋结构，并且在GAAVM中断序列内将GAA替换为GPO或VM替换为PO会降低稳定性。因此，刚性亚氨基酸在中断序列内是不利的，尽管它们对三螺旋本身有良好的稳定作用。这些结果表明，三螺旋甘氨酸-X-Y重复序列中这类中断具有一些非随机结构。
