Frustration, Specificity and Function in the NfkB-IkB System

描述（由申请人提供）：重复蛋白在所有三个门中都有发现，并且存在于所有真核生物蛋白编码序列的约6%中[1]。它们由类似氨基酸延伸的串联阵列组成，这些氨基酸延伸折叠成重复结构基序的细长结构，这些结构基序一个接一个地堆叠，产生延伸的超螺旋结构[2]。重复序列蛋白质由于其固有的一级序列和三维结构的对称性，成为序列-密码-折叠-密码-功能假说的重要模型。蛋白质折叠的能量景观理论是基于最小挫折原则[3]。然而，这一原理并不排除折叠蛋白质中可能存在某种能量挫折。此外，剩余的挫折可能有助于蛋白质在其天然盆地周围的运动，因此剩余的挫折可能是蛋白质功能的基础[4]。最近开发的用于空间定位和量化天然蛋白质结构中存在的能量挫折的方法反映了对蛋白质能量景观的进化约束，并产生了对其生物学功能的有用见解[5]。我们把注意力集中在I：B蛋白上。这些含有锚定重复序列（AR）的蛋白质调节NF-：B转录因子家族的活性，发现NF-：B转录因子家族在诸如癌症、关节炎、哮喘、糖尿病、AIDS和病毒感染的疾病中被错误调节[6]。I：B1的锚定重复区含有6个AR，当不与NF-：B复合时，显示出高度动态特征[7-9]。此外，它已被证明其折叠性能与其功能明显相关[10]。我们设想了一种补充方法，以实现母基金项目3中提出的以下目标：1）表征NF-：B/ I：B1系统中偶联折叠和结合的总体动力学。2)表征NF-：B/ I：B1系统中偶联折叠和结合的残基特异性动力学。这些需要一个共同的理论和实验的努力，汇集计算分析的分布的本地挫折，其影响的折叠过渡（模拟和测量），以及这些如何影响功能结合反应。我们将使用生物信息学方法来表征各种NF-：B和I：B家族成员[5]在自由和结合状态下的挫折模式。我们将使用计算模型来模拟I：B1 - NF-：B的折叠结合反应，使用最近开发的允许能量挫折的方案[11]。我们将设计扰动挫折分布的突变[11]，在实验室中实现这些突变，并通过量化适当的热力学观测值[8，10，12]来分析结构转变和结合性质如何受到影响。这项研究将主要在阿根廷布宜诺斯艾利斯的Quilmes国立大学与Diego Ferreiro博士合作进行，作为NIH资助号P01-GM 071862 -01的扩展。公共卫生相关性：转录因子是控制基因组mRNA合成的蛋白质。在转录因子中，发现NF-：B转录因子家族在诸如癌症、关节炎、哮喘、糖尿病、AIDS和病毒感染的疾病中失调[6]。该家族受称为I：Bs的抑制剂调节，我们已经证明I：B1在与NF -：B结合时折叠[10]。该蛋白质表现出的复杂折叠转变（拟议研究的主题）可能是其生物学功能的关键，因为它们可能与其NF-：B识别和抑制率相关[13]，NF-：B信号传导系统整体行为的关键过程[14]。

DESCRIPTION (provided by applicant): Repeat proteins are found in all three phyla and present in some 6% of all eukaryotic protein coding sequences [1]. They are made up of tandem arrays of similar amino acid stretches that fold up into elongated architectures of repeating structural motifs that stack one upon the next producing extended superhelical structures [2]. Repeat proteins, by virtue of their inherent symmetries both in primary sequence and three dimensional structure, stand as remarkable models where the sequence-codes-folding-codes-function< hypothesis can be quantitatively evaluated. The energy landscape theory of protein folding is based on the principle of minimal frustration< [3]. This principle, however, does not rule out that some energetic frustration may be present in a folded protein. Moreover, the remaining frustration may facilitate motion of the protein around its native basin, and as such the residual frustration may be fundamental to protein function [4]. Recently developed methods for spatially localizing and quantifying the energetic frustration present in native protein structures reflects evolutionary constraints on a proteins< energy landscape and yields useful insights into their biological functions [5]. We focus our attention on the I:B proteins. These ankyrin-repeat (AR) containing proteins regulate the activity of the NF-:B transcription factor family, which is found misregulated in diseases such as cancer, arthritis, asthma, diabetes, AIDS and viral infections [6]. The Ankyrin-repeat region of I:B1 contains 6 ARs and displays a highly dynamic character when not complexed with NF-:B [7-9]. Moreover, it has been shown that its folding properties are clearly related to its function [10]. We envision a complementary approach to the following Aims presented in Project 3 of the parent grant: 1) characterize the overall kinetics of coupled folding and binding in the NF-:B/ I:B1 system. 2) characterize the residue specific dynamics of coupled folding and binding in the NF-:B/ I:B1 system. These require a joint theoretical and experimental effort that brings together computational analysis of the distributions of local frustration, its effect on the folding transitions (both simulated and measured), and how these affect the functional binding reactions. We will use bioinformatic methods to characterize the frustration patterns of various NF-:B and I:B family members [5], both in their free and bound states. We will use computational models to simulate the folding - binding reaction of I:B1 - NF-:B, using recently developed schemes that allow for energetic frustration [11]. We will design mutations that perturb the frustration distributions [11], realize those in the laboratory, and analyze how the structural transitions and the binding properties are affected by means of quantification of the appropriate thermodynamic observables [8,10,12]. This research will be done primarily in Buenos Aires, Argentina, at the Universidad Nacional de Quilmes, in collaboration with Dr Diego Ferreiro, as an extension of NIH Grant No P01-GM071862-01. PUBLIC HEALTH RELEVANCE: Transcription factors are proteins that control the synthesis of mRNA from genomes. Among transcription factors, the NF-:B transcription factor family, is found misregulated in diseases such as cancer, arthritis, asthma, diabetes, AIDS and viral infections [6]. This family is regulated by inhibitors called I:Bs and we have shown that I:B1 ;folds< upon binding to NF -:B [10]. The intricate folding transitions that this protein exhibits, the topic of the proposed research, may be a key to its biological functions, as they are likely to be related to its NF-:B recognition and inhibition rates [13], crucial processes in the overall behavior of the NF-:B signaling system [14].