Shiba Rumi, Umeyama Mika, Tsukasa Sayaka, Kamikubo Hironari, Yamazaki Yoichi, Yamaguchi Mariko, Iwakura Masahiro, Kataoka Mikio
Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), Ikoma, Nara 630-0192, Japan.
Protein Design Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Biophysics (Nagoya-shi). 2011 Jan 19;7:1-10. doi: 10.2142/biophysics.7.1. eCollection 2011.
Decoding sequence information is equivalent to elucidating the design principles of proteins. For this purpose, we conducted systematic alanine insertion analysis to reveal the regions in the primary structure where the sequence continuity cannot be disrupted. We applied this method to dihydrofolate reductase (DHFR), and examined the effects of alanine insertion on structure and the enzymatic activity by solubility assay and trimethoprim resistance, respectively. We revealed that DHFR is composed of "Structure Elements", "Function Elements" and linkers connecting these elements. The "Elements" are defined as regions where the alanine insertion caused DHFR to become unstructured or inactive. Some "Structure Elements" overlap with "Function Elements", indicating that loss of structure leads to loss of function. However, other "Structure Elements" are not "Function Elements", in that alanine insertion mutants of these regions exhibit substrate- or inhibitor-induced folding. There are also some "Function Elements" which are not "Structure Elements"; alanine insertion into these elements deforms the catalytic site topology without the loss of tertiary structure. We hypothesize that these elements are involved essential interactions for structure formation and functional expression. The "Elements" are closely related to the module structure of DHFR. An "Element" belongs to a single module, and a single module is composed of some number of "Elements." We propose that properties of a module are determined by the "Elements" it contains. Systematic alanine insertion analysis is an effective and unique method for deriving the regions of a sequence that are essential for structure formation and functional expression.
解码序列信息等同于阐明蛋白质的设计原则。为此，我们进行了系统的丙氨酸插入分析，以揭示一级结构中序列连续性不能被破坏的区域。我们将此方法应用于二氢叶酸还原酶（DHFR），并分别通过溶解度测定和甲氧苄啶抗性检测了丙氨酸插入对结构和酶活性的影响。我们发现DHFR由“结构元件”、“功能元件”以及连接这些元件的接头组成。“元件”被定义为丙氨酸插入导致DHFR变得无结构或无活性的区域。一些“结构元件”与“功能元件”重叠，表明结构的丧失导致功能的丧失。然而，其他“结构元件”并非“功能元件”，因为这些区域的丙氨酸插入突变体表现出底物或抑制剂诱导的折叠。也有一些“功能元件”并非“结构元件”；将丙氨酸插入这些元件会使催化位点拓扑结构变形而不丧失三级结构。我们推测这些元件参与了结构形成和功能表达所必需的相互作用。“元件”与DHFR的模块结构密切相关。一个“元件”属于单个模块，而单个模块由若干个“元件”组成。我们提出模块的性质由其包含的“元件”决定。系统的丙氨酸插入分析是一种推导序列中对结构形成和功能表达至关重要区域的有效且独特的方法。
