Thermal Unfolding of Proteins Probed at the Single Molecule Level Using Nanopores

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• 作者：Linda Payet ; Marl猫ne Martinho ; Manuela Pastoriza-Gallego ; Jean-Michel Betton ; Lo茂c Auvray ; Juan Pelta ; J茅r么me Math茅
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：May 1, 2012
• 年：2012
• 卷：84
• 期：9
• 页码：4071-4076
• 全文大小：278K
• 年卷期：v.84,no.9(May 1, 2012)
• ISSN：1520-6882

文摘

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and 伪-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 掳C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 掳C) using the bulk and single molecule techniques.