Pastor Isabel, Ferrer Maria L, Lillo M Pilar, Gómez Javier, Mateo C Reyes
Instituto de Biología Molecular y Celular, Universidad Miguel HernAndez, 03202-Elche, Spain.
J Phys Chem B. 2007 Oct 4;111(39):11603-10. doi: 10.1021/jp074790b. Epub 2007 Sep 13.
Proteins entrapped in sol-gel matrices have been extensively studied during the last 15 years, showing that most of them can be encapsulated with retention of their native structure and functionality and with enhanced stability. However, relatively little is known about the structural and dynamical details of the biomolecule-matrix interactions. To achieve this goal, the model protein hen egg white lysozyme (HEWL) has been entrapped in sol-gel matrices prepared from tetraethyl orthosilicate through an alcohol-free sol-gel route, and the photophysical properties of its fluorescent tryptophans have been determined using both steady-state and time-resolved fluorescence techniques. By combining fluorescence spectra, quenching experiments, lifetimes, and time-resolved fluorescence anisotropy measurements, we have obtained information on the structure, dynamics, and solvation properties of the entrapped protein. Our results show that the environment of HEWL within the silica pore as well as its internal dynamics is similar to that in aqueous solution, except that the protein showed no or, depending on conditions, very much slower global motion but retained its internal angularly restricted (hindered) segmental rotation upon entrapment. The experiments carried out at different experimental conditions indicate that, below the isoelectric point of the protein, a strong electrostatic interaction is established between the protein molecule and the negatively charged sol-gel walls, which is ultimately responsible for the total arrest of the overall rotation of the protein, but without significant effect upon its segmental rotational relaxation. The electrostatic nature of the interaction is clearly established since either reducing the positive charge of the protein (by increasing the pH toward its isoelectric point) or increasing the ionic strength of the solution (shielding against the attractive interaction) leads to a situation in which the protein freely rotates within the matrix pore, albeit an order of magnitude more slowly than that in free solution under similar macroscopic solution conditions, and still retains its segmental rotational properties.
在过去15年里，人们对包埋于溶胶-凝胶基质中的蛋白质进行了广泛研究，结果表明大多数蛋白质在包埋后能够保持其天然结构和功能，并且稳定性增强。然而，关于生物分子与基质相互作用的结构和动力学细节，我们了解得还相对较少。为了实现这一目标，我们将模型蛋白鸡蛋清溶菌酶（HEWL）包埋于通过无醇溶胶-凝胶法由正硅酸四乙酯制备的溶胶-凝胶基质中，并使用稳态和时间分辨荧光技术测定了其荧光色氨酸的光物理性质。通过结合荧光光谱、猝灭实验、寿命以及时间分辨荧光各向异性测量，我们获得了关于包埋蛋白的结构、动力学和溶剂化性质的信息。我们的结果表明，二氧化硅孔内HEWL的环境及其内部动力学与水溶液中的情况相似，只是该蛋白在包埋后没有整体运动，或者根据条件，整体运动非常缓慢，但仍保留其内部角度受限（受阻）的片段旋转。在不同实验条件下进行的实验表明，在蛋白质的等电点以下，蛋白质分子与带负电的溶胶-凝胶壁之间会形成强烈的静电相互作用，这最终导致蛋白质整体旋转完全停止，但对其片段旋转弛豫没有显著影响。由于降低蛋白质的正电荷（通过将pH值向其等电点增加）或增加溶液的离子强度（屏蔽吸引相互作用）都会导致蛋白质在基质孔内自由旋转，尽管比在类似宏观溶液条件下的自由溶液中慢一个数量级，但仍保留其片段旋转特性，因此相互作用的静电性质得以明确确立。
