Guttenplan Alexander P M, Young Laurence J, Matak-Vinkovic Dijana, Kaminski Clemens F, Knowles Tuomas P J, Itzhaki Laura S
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
J Nanobiotechnology. 2017 Oct 6;15(1):70. doi: 10.1186/s12951-017-0300-7.
Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled.
Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of "click" reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology.
Although previous studies have produced peptides that can both form amyloid fibrils and undergo "click"-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.
由于蛋白质和肽具有自组装的天然倾向，它们是有机纳米技术的重要组成部分。最近引起关注的一类特殊肽是那些形成淀粉样纤维的肽，因为这种自组装会产生极其坚固、稳定的准一维结构，可用于组织包括蛋白质和寡核苷酸在内的多种货物种类。然而，已经与蛋白质缀合的肽的组装仅限于那些在空间上不干扰组装过程或在常用于组装的苛刻条件下不会错误折叠的货物种类。因此，需要一种通用方法在淀粉样纤维自组装后将蛋白质和其他分子缀合到淀粉样纤维上。
在这里，我们基于转甲状腺素蛋白的TTR105 - 115片段设计了一种淀粉样生成肽，以形成在其表面显示炔烃官能团的纤维，这对于生物正交化学反应很重要。通过Huisgen环加成反应（“点击”反应之一），这些纤维与含叠氮化物的氨基酸以及叠氮化物功能化的染料形成并发生反应。质谱和全内反射荧光光学显微镜用于表明掺入纤维中的肽与叠氮化物反应，同时保持纤维的结构。这些点击功能化的淀粉样纤维在材料和作为生物纳米技术的支架方面有多种潜在用途。
尽管先前的研究已经产生了既能形成淀粉样纤维又能进行“点击”型反应的肽，但这是淀粉样纤维在形成后能够进行此类反应的首个例子。我们的方法具有这样的优势，即自组装在点击功能化之前发生，而不是预功能化的构建块进行自组装。因此，用于功能化纤维的分子本身不必暴露于苛刻的淀粉样形成条件下。这意味着在此过程中可以使用更广泛的蛋白质作为配体。例如，纤维可以用绿色荧光蛋白进行功能化，该蛋白在附着到纤维后仍保留其荧光，而如果将该蛋白暴露于用于聚集的条件下，它会失去荧光。
