Enzymatic Proteolysis of a Surface-Bound α-Helical Polypeptide
Enzymatic Proteolysis of a Surface-Bound α-Helical Polypeptide
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DOI：
10.1021/la8020386
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发表时间：
2008-12-16
期刊：
影响因子：
3.9
通讯作者：
Frank, Curtis W.
中科院分区：
文献类型：
--
作者：
Hardesty, Jasper O.;Cascao-Pereira, Luis;Frank, Curtis W.
在这项工作中，我们研究了酶与模型基板表面的相互作用，使用无标记技术。我们的模型系统是基于丝氨酸蛋白酶（一类酶的蛋白质）和表面结合的多肽底物。虽然以前的研究集中在散装介质的因素，如pH值，离子强度和表面活性剂，这项研究的重点是表面结合基板本身的作用。特别是，我们评估如何基板密度的多肽与α-螺旋二级结构的影响表面反应性。基于表明稳定的α-螺旋可以抵抗酶消化的文献选择α-螺旋二级结构。为了研究表面结合的α-螺旋的蛋白酶抗性，我们设计了α-螺旋多肽（SS-多肽，其中SS =二硫化物），使用它形成不同表面覆盖度的膜，然后测量膜对酶暴露的响应。使用石英晶体微天平与耗散（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，我们的特点是降解的薄膜，以确定如何的表面结合的SS-多肽基板的横向堆积密度影响表面蛋白水解。纯SS-多肽膜的表征表明保持其螺旋结构的螺旋的密集堆积，并且通常垂直于表面取向。我们发现，纯SS-多肽的膜显著抵抗酶消化，而在这种膜中掺入非常少量的稀释剂导致快速消化。部分地，这可能是由于酶需要沿着裂缝内的肽底物沿着结合几种肽以进行消化。只有致密堆积且不允许催化接触多种肽的SS-多肽膜（例如，仅末端肽）对酶促蛋白水解有抗性。
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 alpha-helix, we designed an alpha-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.