Hull James R, Tamura Glen S, Castner David G
National ESCA and Surface Analysis Center for Biomedical Problems, and Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, USA.
Biophys J. 2007 Oct 15;93(8):2852-60. doi: 10.1529/biophysj.107.109819.
Understanding the interactions of adsorbed fibronectin (Fn) with other biomolecules is important for many biomedical applications. Fn is found in almost all body fluids, in the extracellular matrix, and plays a fundamental role in many biological processes. This study found that the structure (conformation, orientation) and reactivity of Fn adsorbed onto mica is dependent on the Fn surface concentration. Atomic force microscopy and x-ray photoelectron spectroscopy were used to determine the surface coverage of adsorbed Fn from isolated molecules at low surface coverage to full monolayers at high surface coverage. Both methods showed that the thickness of Fn film continued to increase after the mica surface was completely covered, consistent with Fn adsorbed in a more upright conformation at the highest surface-Fn concentrations. Time-of-flight secondary ion mass spectrometry showed that relative intensities of both sulfur-containing (cystine, methionine) and hydrophobic (glycine, leucine/isoleucine) amino acids varied with changing Fn surface coverage, indicating that the conformation of adsorbed Fn depended on surface coverage. Single-molecule force spectroscopy with collagen-related peptides immobilized onto the atomic force microscope tip showed that the specific interaction force between the peptide and Fn increases with increasing Fn surface coverage.
了解吸附的纤连蛋白（Fn）与其他生物分子之间的相互作用对于许多生物医学应用而言至关重要。Fn几乎存在于所有体液和细胞外基质中，并且在许多生物过程中发挥着重要作用。本研究发现，吸附在云母上的Fn的结构（构象、取向）和反应性取决于Fn的表面浓度。利用原子力显微镜和X射线光电子能谱来确定从低表面覆盖率下的孤立分子到高表面覆盖率下的完整单分子层吸附Fn的表面覆盖率。两种方法均表明，云母表面完全覆盖后，Fn膜的厚度仍持续增加，这与在最高表面Fn浓度下以更直立构象吸附的Fn一致。飞行时间二次离子质谱表明，含硫（胱氨酸、蛋氨酸）和疏水（甘氨酸、亮氨酸/异亮氨酸）氨基酸的相对强度随Fn表面覆盖率的变化而变化，表明吸附的Fn的构象取决于表面覆盖率。将与胶原蛋白相关的肽固定在原子力显微镜尖端进行的单分子力谱显示，肽与Fn之间的特异性相互作用力随Fn表面覆盖率的增加而增大。
