Caulobacter S-layer structure and regulation

我们研究了新月藻的蛋白表面(S)层的生物发生。这种二维晶格包裹着细菌，可以与许多细菌和古生物的S层相媲美。这是为数不多的由1型蛋白分泌输出的S层。 意义：1)S-层次感。很少有人对其进行详细的分子研究，对其结构原理也知之甚少。2)1型分泌物。革兰氏(-)细菌，特别是病原体的重要分泌机制，但很少有研究机制的细节3)生物技术。了解Caulbacter S层结构对于设计和展示作为人类治疗剂的异源蛋白具有重要意义。 具体目标和重点意义有四个方面： 1)S--层次型结构。S层的原子分辨率具有挑战性，因为很难结晶出自组装的蛋白质。我们将蛋白质分解成它的区域(附着、结晶、分泌信号)，并通过合作，结晶和解决结构。每个区域都解决了重要的问题：a)附着结构域是一种特殊的凝集素，在Gram(-)中几乎没有表面附着的范例，b)1型C末端分泌信号知之甚少，没有解决的结构，c)结晶域的RTX基序对任何1型分泌蛋白的作用都不清楚。 2)分泌/翻译调控。分泌处于极端水平，但当被阻断时，会出现强烈的转录后下调。我们将对翻译起始区进行定点突变，比较S层截短的表达，并检测小RNA以寻找控制的基础。 3)附着型内毒素。S层通过脂多糖附着。我们将评估O-侧链的组成，这是一种明显独特的修饰的过氧胺残基的排列，并将其与附着结构域共结晶。 4)S层相关蛋白水解酶。如果折叠不正确，这种酶会导致S层的裂解，并导致外来插入物内的定点裂解。我们将学习它是如何附着在细胞表面并对S层进行编辑的。

We study biogenesis of the protein surface (S)-layer of Caulobacter crescentus. This 2-D crystalline lattice encases the bacterium and is comparable to S-layers of many bacteria and Archaea. This S-layer is one of few exported by type 1 protein secretion. Significance: 1) S-layers. Few are studied in molecular detail and structural principles are poorly understood. 2) Type 1 secretion. An important secretion mechanism for Gram (-) bacteria, especially pathogens, yet few study mechanistic details 3) Biotechnology. Understanding Caulobacter S-layer structure has utility in design and display of heterologous proteins used as human therapeutic agents. The Specific Objectives and Focused Significance are in 4 areas: 1) S-layer structure. Atomic resolution of S-layers is challenging because of difficulties in crystallizing a self-assembling protein. We will dissect the protein into its domains (attachment, crystallization, secretion signal), and by collaboration, crystallize and solve structures. Each region addresses important issues: a) the attachment domain is a specialized lectin and there are few paradigms for surface attachment in Gram (-), b) Type 1 C-terminal secretion signals are poorly understood, with no solved structures and c) the role of the RTX motif of the crystallization domain is not understood for any type 1 secreted protein. 2) Secretion/translation Regulation. Secretion is at extreme levels, but when blocked there is strong posttranscriptional downregulation. We will do site-directed mutagenesis of mRNA translation initiation regions, compare S-layer truncates for expression and examine small RNAs to find the basis of control. 3) Attachment LPS. The S-layer attaches via LPS. We will assess O-side chain composition, an apparently unique arrangement of modified perosamine residues and co-crystallize it with the attachment domain. 4) S-layer associated protease. This protease causes cleavage of S-layer if not properly folded and site-specific cleavage within foreign inserts. We will learn how it attaches to the cell surface and "edits" S-layer.