Garcia Tzintzuni I, Oberhauser Andres F, Braun Werner
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Proteins. 2009 May 15;75(3):706-18. doi: 10.1002/prot.22281.
The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single-molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I-band of a sarcomere is composed of about 40 Ig-domains and is exposed to force under normal physiological conditions and connects the free-hanging ends of the myosin filaments to the Z-disc. Recent single-molecule force spectroscopy data show a mechanical hierarchy in the I-band domains. Domains near the C-terminus in this region unfold at forces two to three times greater than domains near the beginning of the I-band. Though all of these Ig-domains are thought to share a fold and topology common to members of the Ig-like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I-band Ig domain to specific, conserved physical-chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into "strong" and "weak" families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the beta-sandwich fold, and force sensitive residues are not only confined to the A'-G region.
决定蛋白质折叠机械稳定性的机制仍然难以捉摸。我们对这些机制的理解对于生物工程研究以及更好地理解和最终治疗影响机械重要蛋白质（如肌联蛋白）的致病突变都至关重要。我们提出了一种新方法，用于分析来自巨肌蛋白肌联蛋白不同结构域的单分子力谱数据。在肌节I带中发现的肌联蛋白区域由大约40个免疫球蛋白（Ig）结构域组成，在正常生理条件下会受到力的作用，并将肌球蛋白丝的自由悬挂端连接到Z盘。最近的单分子力谱数据显示了I带结构域中的机械层次结构。该区域C末端附近的结构域在比I带起始附近的结构域大两到三倍的力作用下展开。尽管所有这些Ig结构域都被认为具有Ig样折叠家族成员共有的折叠和拓扑结构，但相邻结构域的序列差异很大，平均序列同一性仅为25%。在本研究中，我们研究了每个I带Ig结构域的这些独特机械稳定性与相关Ig结构域中氨基酸序列特定的、保守的物理化学性质之间的关系。我们发现，每个单独的肌联蛋白Ig结构域的序列高度保守，在如人类、鸡和斑马鱼等不同物种中平均序列同一性为79%。这表明每个结构域的机械性质得到了很好的保留，并与其在肌联蛋白分子中的独特位置相适应。我们使用PCPMer软件来确定按展开力分组为“强”和“弱”家族的肌联蛋白Ig结构域中氨基酸性质的保守性。我们发现每个家族都有两个独特的基序，它们可能在决定这些Ig结构域的机械性质方面发挥了一定作用。对单个残基性质的详细统计分析揭示了几个在强家族和弱家族中显示出不同保守性质的位置。与之前的研究不同，我们发现有证据表明Ig结构域的机械稳定性是由散布在β-三明治折叠中的几个残基决定的，并且力敏感残基不仅局限于A'-G区域。
