González-Montalbán Nuria, García-Fruitós Elena, Ventura Salvador, Arís Anna, Villaverde Antonio
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Microb Cell Fact. 2006 Aug 7;5:26. doi: 10.1186/1475-2859-5-26.
The molecular mechanics of inclusion body formation is still far from being completely understood, specially regarding the occurrence of properly folded, protein species that exhibit natural biological activities. We have here comparatively explored thermally promoted, in vivo protein aggregation and the formation of bacterial inclusion bodies, from both structural and functional sides. Also, the status of the soluble and insoluble protein versions in both aggregation systems have been examined as well as the role of the main molecular chaperones GroEL and DnaK in the conformational quality of the target polypeptide.
While thermal denaturation results in the formation of heterogeneous aggregates that are rather stable in composition, protein deposition as inclusion bodies renders homogenous but strongly evolving structures, which are progressively enriched in the main protein species while gaining native-like structure. Although both type of aggregates display common features, inclusion body formation but not thermal-induced aggregation involves deposition of functional polypeptides that confer biological activity to such particles, at expenses of the average conformational quality of the protein population remaining in the soluble cell fraction. In absence of DnaK, however, the activity and conformational nativeness of inclusion body proteins are dramatically impaired while the soluble protein version gains specific activity.
The chaperone DnaK controls the fractioning of active protein between soluble and insoluble cell fractions in inclusion body-forming cells but not during thermally-driven protein aggregation. This cell protein, probably through diverse activities, is responsible for the occurrence and enrichment in inclusion bodies of native-like, functional polypeptides, that are much less represented in other kind of protein aggregates.
包涵体形成的分子机制仍远未被完全理解，特别是关于具有天然生物活性的正确折叠蛋白质种类的出现情况。我们在此从结构和功能两方面对热促进的体内蛋白质聚集以及细菌包涵体的形成进行了比较研究。此外，还研究了两种聚集系统中可溶性和不溶性蛋白质形式的状态，以及主要分子伴侣GroEL和DnaK在靶多肽构象质量方面的作用。
热变性导致形成组成相当稳定的异质聚集体，而作为包涵体的蛋白质沉积产生均匀但结构不断演变的结构，这些结构在主要蛋白质种类中逐渐富集，同时获得类似天然的结构。尽管两种聚集体都有共同特征，但包涵体形成而非热诱导聚集涉及功能性多肽的沉积，这些多肽赋予此类颗粒生物活性，代价是留在可溶性细胞部分的蛋白质群体的平均构象质量。然而，在没有DnaK的情况下，包涵体蛋白的活性和构象天然性会显著受损，而可溶性蛋白形式则获得比活性。
伴侣蛋白DnaK控制形成包涵体的细胞中活性蛋白在可溶性和不溶性细胞部分之间的分配，但在热驱动的蛋白质聚集过程中不起作用。这种细胞蛋白可能通过多种活性，导致类似天然的功能性多肽在包涵体中出现和富集，而在其他类型的蛋白质聚集体中则较少出现。
