Li Yaoxin, Ogorzalek Tadeusz L, Wei Shuai, Zhang Xiaoxian, Yang Pei, Jasensky Joshua, Brooks Charles L, Marsh E Neil G, Chen Zhan
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Phys Chem Chem Phys. 2018 Jan 3;20(2):1021-1029. doi: 10.1039/c7cp06063g.
Tethering peptides and proteins to abiotic surfaces has the potential to create biomolecule-functionalized surfaces with useful properties. Commonly used methods of immobilization lack control over the orientation in which biological molecules are covalently or physically bound to the surface, leading to sub-optimal materials. Here we use an engineered beta-galactosidase that can be chemically immobilized on a surface with a well-defined orientation through unique surface-accessible cysteine residues. A combined study using sum frequency generation (SFG) vibrational spectroscopy and coarse grained molecular dynamics (MD) simulations was performed to determine the effects of enzyme immobilization site and abiotic surface chemistry on enzyme surface orientation, surface coverage, and catalytic activity. Two beta-galactosidase variants that were immobilized through cysteine introduced at positions 227 and 308 were studied. In both cases, when the abiotic surface was made more hydrophilic, the enzyme surface coverage decreased, but the activity increased. MD simulations indicated that this is due to the weakened interactions between the immobilized enzyme and the more hydrophilic surface. These studies provide improved understanding of how enzyme-surface interactions can be optimized to maximize the catalytic activity of surface tethered enzymes.
将肽和蛋白质连接到非生物表面有可能创造出具有有用特性的生物分子功能化表面。常用的固定方法无法控制生物分子与表面共价或物理结合的方向，导致材料性能欠佳。在此，我们使用一种经过工程改造的β-半乳糖苷酶，它可以通过独特的可接近表面的半胱氨酸残基以明确的方向化学固定在表面。我们进行了一项结合和频振动光谱（SFG）与粗粒度分子动力学（MD）模拟的研究，以确定酶固定位点和非生物表面化学对酶表面方向、表面覆盖率和催化活性的影响。研究了通过在227位和308位引入半胱氨酸而固定的两种β-半乳糖苷酶变体。在这两种情况下，当非生物表面变得更亲水时，酶的表面覆盖率降低，但活性增加。MD模拟表明，这是由于固定化酶与更亲水表面之间的相互作用减弱所致。这些研究有助于更好地理解如何优化酶-表面相互作用，以最大限度地提高表面固定化酶的催化活性。
