Departament d'Enginyeria Química and Barcelona Research Center for Multiscale Science and Engineering, EEBE, Universitat Politècnica de Catalunya, C/ Eduard Maristany 10-14, Barcelona 08019, Spain.
Department of Bioengineering, iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais 1, Lisboa 1049-001, Portugal.
Langmuir. 2022 Oct 25;38(42):12905-12914. doi: 10.1021/acs.langmuir.2c02010. Epub 2022 Oct 13.
CREKA [Cys-Arg-(Me)Glu-Lys-Ala, where (Me)Glu refers to -methyl-Glu], an anti-cancer pentapeptide that induces prostate tumor necrosis and significant reduction in tumor growth, was engineered to increase the resistance to endogenous proteases of its parent peptide, CREKA (Cys-Arg-Glu-Lys-Ala). Considering their high tendency to aggregate, the self-assembly of CREKA and CREKA into well-defined and ordered structures has been examined as a function of peptide concentration and pH. Spectroscopic studies and atomistic molecular dynamics simulations reveal significant differences between the secondary structures of CREKA and CREKA. Thus, the restrictions imposed by the (Me)Glu residue reduce the conformational variability of CREKA with respect to CREKA, which significantly affects the formation of well-defined and ordered self-assembly morphologies. Aggregates with poorly defined morphology are obtained from solutions with low and moderate CREKA concentrations at pH 4, whereas well-defined dendritic microstructures with fractal geometry are obtained from CREKA solutions with similar peptide concentrations at pH 4 and 7. The formation of dendritic structures is proposed to follow a two-step mechanism: (1) pseudo-spherical particles are pre-nucleated through a diffusion-limited aggregation process, pre-defining the dendritic geometry, and (2) such pre-nucleated structures coalesce by incorporating conformationally restrained CREKA molecules from the solution to their surfaces, forming a continuous dendritic structure. Instead, no regular assembly is obtained from solutions with high peptide concentrations, as their dynamics is dominated by strong repulsive peptide-peptide electrostatic interactions, and from solutions at pH 10, in which the total peptide charge is zero. Overall, results demonstrate that dendritic structures are only obtained when the molecular charge of CREKA, which is controlled through the pH, favors kinetics over thermodynamics during the self-assembly process.
CREKA [半胱氨酸-精氨酸-（甲硫基）谷氨酸-赖氨酸-丙氨酸，其中（甲硫基）Glu 指 -甲基-Glu] 是一种抗癌五肽，可诱导前列腺肿瘤坏死并显著减少肿瘤生长，其被设计为提高其母体肽 CREKA（半胱氨酸-精氨酸-谷氨酸-赖氨酸-丙氨酸）对内源性蛋白酶的抗性。考虑到它们高度聚集的倾向，已经研究了 CREKA 和 CREKA 自组装成明确定义和有序结构的情况，作为肽浓度和 pH 的函数。光谱研究和原子分子动力学模拟揭示了 CREKA 和 CREKA 之间二级结构的显著差异。因此，（甲硫基）Glu 残基的限制降低了 CREKA 相对于 CREKA 的构象可变性，这对明确定义和有序自组装形态的形成有很大影响。在 pH4 时，低浓度和中等浓度的 CREKA 溶液中得到形态不规则的聚集体，而在 pH4 和 7 时，具有分形几何形状的规则树枝状微结构则从具有相似肽浓度的 CREKA 溶液中获得。提出树枝状结构的形成遵循两步机制：（1）通过扩散限制聚集过程预成核形成准球形颗粒，预定义树枝状几何形状，（2）通过从溶液中掺入构象受限的 CREKA 分子到其表面，使预成核结构合并，形成连续的树枝状结构。相反，在高浓度肽溶液中没有得到规则的组装，因为其动力学受强的肽-肽静电排斥相互作用支配，在 pH10 的溶液中，总肽电荷为零。总体而言，结果表明，只有当 CREKA 的分子电荷，即通过 pH 控制的电荷，在自组装过程中有利于动力学而不是热力学时，才会得到树枝状结构。
