Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, Maryland 21201, USA.
Biochemistry. 2009 Dec 29;48(51):12191-201. doi: 10.1021/bi901640e.
Our recent study established the NMR structure of the recombinant bAalpha406-483 fragment corresponding to the NH(2)-terminal half of the bovine fibrinogen alphaC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the study presented here were to determine the structure and self-association of the full-length human fibrinogen alphaC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAalpha425-503, homologous to bovine bAalpha406-483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAalpha406-483. We also prepared recombinant hAalpha392-610 and bAalpha374-568 fragments corresponding to the full-length human and bovine alphaC-domains, respectively, and tested their structure, stability, and ability to self-associate. Size-exclusion chromatography revealed that both fragments form reversible oligomers in a concentration-dependent manner. Their oligomerization was confirmed in sedimentation equilibrium experiments, which also established the self-association affinities of these fragments and revealed that the addition of each monomer to assembling alphaC-oligomers substantially increases the stabilizing free energy. In agreement, unfolding experiments monitored by CD established that self-association of both fragments results in a significant increase in their thermal stability. Analysis of CD spectra of both fragments revealed that alphaC self-association results in an increase in the level of regular structure, implying that the COOH-terminal half of the alphaC-domain adopts an ordered conformation in alphaC-oligomers and that this domain contains two independently folded subdomains. Altogether, these data further clarify the structure of the human and bovine alphaC-domains and the molecular mechanism of their self-association into alphaC-polymers in fibrin.
我们最近的研究建立了与牛纤维蛋白原αC 结构域 N 端一半相对应的重组 bAalpha406-483 片段的 NMR 结构，并揭示了该片段在浓度增加时形成寡聚物（自身聚集）。本研究的主要目标是确定全长人纤维蛋白原αC 结构域的结构和自身聚集。为了实现这些目标，我们制备了与牛 bAalpha406-483 同源的重组人片段 hAalpha425-503，并通过 NMR、CD 和凝胶过滤色谱证明其整体折叠和形成寡聚物的能力与 bAalpha406-483 相似。我们还制备了重组 hAalpha392-610 和 bAalpha374-568 片段，分别对应全长人源和牛源的αC 结构域，并测试了它们的结构、稳定性和自身聚集能力。凝胶过滤色谱显示，这两个片段都以浓度依赖的方式形成可逆的寡聚物。在沉降平衡实验中证实了它们的寡聚化，该实验还确定了这些片段的自缔合亲和力，并揭示了每个单体加入组装的αC 寡聚体可显著增加稳定自由能。一致的是，通过 CD 监测的解折叠实验表明，这两个片段的自身聚集导致其热稳定性显著增加。对这两个片段的 CD 光谱进行分析表明，αC 自身聚集导致规则结构水平增加，这意味着αC 结构域的 COOH 端在αC 寡聚体中采用有序构象，并且该结构域包含两个独立折叠的亚结构域。总之，这些数据进一步阐明了人源和牛源αC 结构域的结构以及它们自身聚集形成纤维蛋白中αC 聚合物的分子机制。
