CRYSTALLOGRAPHIC STUDIES OF FRAGMENTS FROM REGULATED MYOSIN

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们收集了扇贝肌球蛋白头部和尾部的晶体数据。在我们最近对扇贝肌球蛋白头部（S1）的肌动蛋白分离、内部不耦合状态的高分辨率晶体结构的研究（Himmel等人，在制备中；在我们2001年的国际象棋进展报告中报道）之后，我们现在已经获得了前冲程或启动构象中S1的2.6A分辨率结构（Gourinath等人，在制备中）。这两种结构在子域相互作用中差异很大，对它们的比较将提供关于不同收缩周期状态下连杆灵活性的关键信息。到目前为止，还没有关于肌凝蛋白尾部任何部分的详细结构的报道。利用在CHESS上以2.6A分辨率收集的数据，我们正在确定扇贝肌凝蛋白尾部50个残基长的片段的结构，该片段位于肌凝蛋白头部附近。尾巴的这个区域对肌球蛋白分子的调控特性至关重要（Li等人，在制备中）。基于过去几年在国际象棋收集的数据，我们还发表了原肌球蛋白和纤维蛋白原关键片段的晶体结构报告。在我们对原肌球蛋白n端片段的研究之后（Brown et al., 2001），我们现在已经建立了横纹肌原肌球蛋白的31个残基c端片段的结构，它显示出一种不寻常的四边形构象。这些结果揭示了肌钙蛋白T的特异性识别位点，并阐明了横纹肌独特调节机制的物理基础（Li et al.， PNAS, in press）。在我们努力了解血液凝固的分子基础的同时，我们也研究了纤维蛋白原的晶体结构。我们之前报道了牛纤维蛋白原285 kDa主链的4A结构（Brown et al., 2000），现在已经获得了分子中心区域的1.4A分辨率结构（Madrazo et al., 2001）。这种结构具有显著的二聚体界面，这是以前无法可视化的。综上所述，这些结果提高了我们对纤维蛋白原凝块分子组装的理解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have collected crystal data from both the head and tail regions of scallop myosin. Following our recent study on the high-resolution crystal structure of the actin-detached, internally-uncoupled state of the scallop myosin head (S1) (Himmel, et al., in preparation; reported in our CHESS Progress Report of 2001), we have now obtained a 2.6A resolution structure of S1 in the pre-power stroke or primed conformation (Gourinath, et al., in preparation). Comparison of these two structures, which differ greatly in subdomain interactions, will provide key information on the flexibility of linkages in different states of the contractile cycle. Until now, there has not been a detailed structure reported for any part of the tail of myosin. Using data collected to 2.6A resolution at CHESS, we are determining the structure of a 50-residue-long segment of the scallop myosin tail, located adjacent to the myosin head. This region of the tail is critical for the regulatory properties of the myosin molecule (Li et al., in preparation). Based on data collected at CHESS during the past few years, we have also published reports on crystal structures of key fragments of tropomyosin and fibrinogen. Following our work on an N-terminal fragment of tropomyosin (Brown et al., 2001), we have now established the structure of a 31-residue C-terminal segment of striated-muscle tropomyosin, which shows an unusual splayed conformation. These results reveal a specific recognition site for troponin T and clarify the physical basis for the unique regulatory mechanism of striated muscles (Li et al., PNAS, in press). In our efforts to understand the molecular basis of blood clotting, we have also pursued crystal structures of fibrinogen. We previously reported the 4A structure of the 285 kDa backbone of bovine fibrinogen (Brown et al., 2000), and have now achieved a 1.4A resolution structure of the central domain of the molecule (Madrazo et al., 2001). This structure has a remarkable dimeric interface, which could not previously be visualized. Taken together, these results have improved our understanding of the assembly of the molecule into the fibrinogen clot.