Hardesty Jasper O, Cascão-Pereira Luis, Kellis James T, Robertson Channing R, Frank Curtis W
Biochemistry Department, Genencor International, Palo Alto, California 94304, USA.
Langmuir. 2008 Dec 16;24(24):13944-56. doi: 10.1021/la8020386.
In this work, we studied the interactions of enzymes with model substrate surfaces using label-free techniques. Our model system was based on serine proteases (a class of enzymes that digests proteins) and surface-bound polypeptide substrates. While previous studies have focused on bulk media factors such as pH, ionic strength, and surfactants, this study focuses on the role of the surface-bound substrate itself. In particular, we assess how the substrate density of a polypeptide with an alpha-helical secondary structure influences surface reactivity. An alpha-helical secondary structure was chosen based on literature indicating that stable alpha-helices can resist enzymatic digestion. To investigate the protease resistance of a surface-bound a-helix, we designed an a-helical polypeptide (SS-polypeptide, where SS = disulfide), used it to form films of varying surface coverage and then measured responses of the films to enzymatic exposure. Using quartz-crystal microbalance with dissipation (QCM-D), angle-resolved X-ray photoelectron spectroscopy (AR-XPS), grazing-angle infrared spectroscopy (GAIRS), and other techniques, we characterized the degradation of films to determine how the lateral packing density of the surface-bound SS-polypeptide substrate affected surface proteolysis. Characterization of pure SS-polypeptide films indicated dense packing of helices that maintained their helical structure and were generally oriented normal to the surface. We found that films of pure SS-polypeptide significantly resisted enzymatic digestion, while incorporation of very minor amounts of a diluent in such films resulted in rapid digestion. In part, this may be due to the need for the enzyme to bind several peptides along the peptide substrate within the cleft for digestion to occur. Only SS-polypeptide films that were densely packed and did not permit catalytic access to multiple peptides (e.g., terminal peptides only) were resistant to enzymatic proteolysis.
在这项工作中，我们使用无标记技术研究了酶与模型底物表面的相互作用。我们的模型系统基于丝氨酸蛋白酶（一类消化蛋白质的酶）和表面结合的多肽底物。虽然先前的研究集中在诸如pH值、离子强度和表面活性剂等本体介质因素上，但本研究聚焦于表面结合底物本身的作用。具体而言，我们评估了具有α-螺旋二级结构的多肽的底物密度如何影响表面反应性。选择α-螺旋二级结构是基于文献表明稳定的α-螺旋可以抵抗酶促消化。为了研究表面结合的α-螺旋的蛋白酶抗性，我们设计了一种α-螺旋多肽（SS-多肽，其中SS = 二硫键），用它形成不同表面覆盖率的薄膜，然后测量薄膜对酶暴露的响应。使用具有耗散功能的石英晶体微天平（QCM-D）、角分辨X射线光电子能谱（AR-XPS）、掠角红外光谱（GAIRS）和其他技术，我们对薄膜的降解进行了表征，以确定表面结合的SS-多肽底物的横向堆积密度如何影响表面蛋白水解。纯SS-多肽薄膜的表征表明螺旋紧密堆积，保持其螺旋结构，并且通常垂直于表面取向。我们发现纯SS-多肽薄膜显著抵抗酶促消化，而在这种薄膜中加入极少量的稀释剂会导致快速消化。部分原因可能是酶需要沿着肽底物在裂隙内结合多个肽才能发生消化。只有紧密堆积且不允许催化作用于多个肽（例如仅末端肽）的SS-多肽薄膜才对酶促蛋白水解具有抗性。
