期刊
ANALYTICAL CHEMISTRY
卷 87, 期 3, 页码 1821-1828出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac503845f
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资金
- Max Planck Society
- Max Planck Institute for Biophysical Chemistry
- Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain
- DFG [SFB860]
- Gottingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences
Protein aggregation is a widely studied phenomenon that is associated with many human diseases and with the degradation of biotechnological products. Here, we establish a new label-free method for characterizing the aggregation kinetics of proteins into amyloid fibrils by suspended microchannel resonators (SMR). SMR devices are unique in their ability to provide mass-based measurements under reaction-limited conditions in a 10 pL volume. To demonstrate the method, insulin seed fibrils of defined length, characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM), were covalently immobilized inside microchannels embedded within a micromechanical resonator, and the elongation of these fibrils under a continuous flow of monomer solution (rate similar to 1 nL/s) was measured by monitoring the resonance frequency shift. The kinetics for concentrations below similar to 0.6 mg/mL fits well with an irreversible bimolecular binding model with the rate constant kon = (1.2 +/- 0.1) x103 M-1 s(-1). Rate saturation occurred at higher concentrations. The nonlinear on-rate for monomer concentrations from 0 to 6 mg/mL and for temperatures from 20 to 42 degrees C fit well globally with an energy landscape model characterized by a single activation barrier. Finally, elongation rates were studied under different solution conditions and in the presence of a small molecule inhibitor of amyloid growth. Due to the low volume requirements, high precision, and speed of SMR measurements, the method may become a valuable new tool in the screening for inhibitors and the study of fundamental biophysical mechanisms of protein aggregation processes.
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