Investigation of mechanical properties of insulin crystals by atomic force microscopy
Investigation of mechanical properties of insulin crystals by atomic force microscopy
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DOI：
10.1021/la7018605
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发表时间：
2008-02-05
期刊：
影响因子：
3.9
通讯作者：
Akhremitchev, Boris B.
中科院分区：
文献类型：
--
作者：
Guo, Senli;Akhremitchev, Boris B.
蛋白质晶体和聚集体的机械性能取决于单个蛋白质分子的构象和结构特性以及固体材料内的堆积密度和结构。建立了一种基于原子力显微镜（AFM）的方法，通过纳米压痕测量小蛋白质晶体的弹性模量，并将其应用于胰岛素晶体的弹性测量。沉积在云母衬底上的晶体的顶面被识别为（001）面。胰岛素晶体在压缩循环中表现出近乎弹性的反应。测得的顶面弹性模量呈不对称分布，且宽度显著。这个宽度与挠度灵敏度的不确定度有关。采用考虑灵敏度分布的模型对弹性模量进行校正。用三种不同的AFM探针对不同位置的几个晶体在水缓冲液中进行测量，平均弹性模量为164 +/- 10 MPa。这个值接近于其他蛋白质晶体的静态弹性模量，这些蛋白质晶体通常在100 MPa到1 GPa的范围内测量。胰岛素晶体的测量模量介于之前在两个正交方向上测量的胰岛素淀粉样原纤维弹性模量之间（垂直于原纤维轴方向压缩原纤维测得14 MPa的模量，沿原纤维轴方向测得3.3 GPa的模量）。这一比较表明，原纤维在垂直于原纤维轴方向上呈非均匀结构，淀粉样蛋白原纤维核心的堆积密度高于胰岛素晶体的平均堆积密度。胰岛素晶体的机械磨损是在AFM测量中检测到的。在胰岛素晶体的纳米压痕实验中，AFM尖端的压缩载荷（约为1 nN，对应于约5 MPa的压力）偶尔会顺序地从胰岛素晶体的顶部或第二层去除蛋白质分子。提出了这种表面损伤的分子模型。此外，在交流模式成像过程中观察到多层分子的去除。移除的层数取决于扫描大小。
Mechanical properties of protein crystals and aggregates depend on the conformational and structural properties of individual protein molecules as well as on the packing density and structure within solid materials. An atomic force microscopy (AFM)-based approach is developed to measure the elastic modulus of small protein crystals by nanoindentation and is applied to measure the elasticity of insulin crystals. The top face of the crystals deposited on mica substrates is identified as the (001) face. Insulin crystals exhibit a nearly elastic response-during the compression cycle. The elastic modulus measured on the top face has asymmetric distribution with a significant width. This width is related to the uncertainty in the deflection sensitivity. A model that takes into account the distribution of the sensitivity values is used to correct the elastic modulus. Measurements performed in aqueous buffer on several crystals at different locations with three different AFM probes give a mean elastic modulus of 164 +/- 10 MPa. This value is close to the static elastic moduli of other protein crystals measured by different techniques that are usually measured in the range from 100 MPa to 1 GPa. The measured modulus of insulin crystals falls between the elastic modulus values of insulin amyloid fibrils measured previously at two orthogonal directions (a modulus of 14 MPa was measured by compressing the fibril in the direction perpendicular to the fibril axis, and a modulus of 3.3 GPa was measured in the direction along the fibril axis). This comparison indicates the heterogeneous structure of fibrils in the direction perpendicular to the fibril axis, with a packing density of the amyloid fibril core that is higher than the average packing density in insulin crystals. The mechanical wear of insulin crystals is detected during AFM measurements. In nanoindentation experiments on insulin crystal, the compressive load by the AFM tip (similar to 1 nN, corresponding to a pressure of around 5 MPa) occasionally removes protein molecules from the top or the second top layer of insulin crystal in a sequential manner. The molecular model of this surface damage is proposed. In addition, the removal of the multiple layers of molecules is observed during the AC-mode imaging in aqueous buffer. The number of removed layers depends on the scan size.