Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Biophys J. 2011 Jan 19;100(2):489-97. doi: 10.1016/j.bpj.2010.12.3689.
We used an atomic force microscope to study the mechanism underlying the translocation of substrate molecules inside the proteasome. Our specific experimental setup allowed us to measure interaction forces between the 20S proteasome and its substrates. The substrate (β-casein) was covalently bound either via a thiol-Au bond or by a PEG-based binding procedure to the atomic force microscope cantilever tip and offered as bait to proteasomes from Methanosarcina mazei. The proteasomes were immobilized densely in an upright orientation on mica, which made their upper pores accessible for substrates to enter. Besides performing conventional single-molecule force spectroscopy experiments, we developed a three-step procedure that allows the detection of specific proteasome-substrate single-molecule events without tip-sample contact. Using the active 20S wild type and an inactive active-site mutant, as well as two casein mutants bound with opposite termini to the microscope tip, we detected no directional preference of the proteasome-substrate interactions. By comparing the distribution of the measured forces for the proteasome-substrate interactions, were observed that a significant proportion of interaction events occurred at higher forces for the active versus the inactive proteasome. These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome.
我们使用原子力显微镜研究了底物分子在蛋白酶体内部易位的机制。我们的具体实验设置允许我们测量 20S 蛋白酶体与其底物之间的相互作用力。底物（β-酪蛋白）通过硫醇-Au 键或基于 PEG 的结合程序共价结合到原子力显微镜悬臂尖端，并作为诱饵提供给产甲烷八叠球菌的蛋白酶体。蛋白酶体在云母上密集地垂直固定，使其上部孔道可供底物进入。除了进行传统的单分子力谱实验外，我们还开发了一种三步程序，该程序允许在不接触针尖的情况下检测特定的蛋白酶体-底物单分子事件。使用活性 20S 野生型和失活的活性位点突变体，以及与显微镜尖端结合的相反末端的两种酪蛋白突变体，我们没有检测到蛋白酶体-底物相互作用的方向偏好。通过比较蛋白酶体-底物相互作用的测量力分布，我们观察到对于活性蛋白酶体，与失活蛋白酶体相比，相当一部分相互作用事件发生在更高的力下。这些力可以归因于底物在通往位于蛋白酶体内部深处的活性部位的易位过程中。
