| Summary: | 博士 === 中興大學 === 生物科技學研究所 === 99 === The study focus on the characterization and nanomechanics of non-mechanical proteins in order to expand the toolbox of elastomeric proteins, which can be used for designing or being modules of protein-based building blocks that are incorporated into multifunctional nano-structured assembles. In detail, this research aims to: (1) the methodology established of measuring mechanical stability of non-mechanical proteins. We genetically construct the chimera polyprotein system with reference proteins, which are necessary to identify the single molecule force - extension recordings probed by AFM. (2) We use AFM-based single molecule force spectroscopy to directly measure mechanical unfolding forces of SNase alone and in the complex with (pdTp)-Ca+2 and demonstrated that the insertion of the nucleotide inhibitor significantly enhances the mechanical stability of the enzyme. (3) We use AFM-based single molecule force spectroscopy to directly measure mechanical unfolding forces of cytochrome b562 in the different oxidation states, and demonstrated that the reduction of iron center can enhance the mechanical stability of cytochrome b562.
The methodology established of measuring mechanical stability of non-mechanical proteins. The current challenge on AFM based single - molecule force spectroscopy has been to identify single-molecule AFM force-extension recordings and reduce the background signal masked by the non-specifically interactions between tip and substrate. The proteins usually were immobilized on substrate and picked up by the tip non-specifically. Therefore, it recommends that a longer protein in size is necessary in AFM pulling experiments. Otherwise, the build-in mechanically reference proteins can be very helpful to identify the single molecule records due to its well-defined mechanical properties. Thus, we combined genetic and molecular approaches to fill this lacuna. In the study, we genetically engineered a chimera polyprotein system, in which interest are flanked by I27 domains of titin. I27 domains serve here as molecular handles allowing to apply stretching forces to the N and C termini of protein and also serve as a mechanical reference allowing to identify single-molecule AFM force-extension recordings.
The characterization and nanomechanics of Staphylococcal nuclease. Staphylococcal nuclease (SNase) catalyzes the hydrolysis of DNA and RNA in a calcium-dependent fashion. We used AFM-based single-molecule force spectroscopy to investigate the mechanical stability of SNase alone and in its complex with an SNase inhibitor, deoxythymidine 30,50-bisphosphate. We found that the enzyme unfolds in an all-or-none fashion at ~26 pN. Upon binding to the inhibitor, the mechanical unfolding forces of the enzyme-inhibitor complex increase to ~50 pN. This inhibitor-induced increase in the mechanical stability of the enzyme is consistent with the increased thermodynamical stability of the complex over that of SNase. Because of its strong mechanical response to inhibitor binding, SNase, a model protein folding system, offers a unique opportunity for studying the relationship between enzyme mechanics and catalysis.
The characterization and nanomechanics of E.coli cytochrome b562. Heme redox proteins offer a unique opportunity to examine the effect of redox reactions on the mechanical stability of heme proteins, which can be probed by SMFS. Here, we used SMFS to directly measure the effect of heme and its oxidation state on the mechanical properties of cytochrome b562 (cyt b562). Our results show that the reduced cytochrome b562 has indeed a higher mechanical stability as compared to the oxidized cytochrome. The average unfolding force ﹤Funfolding ﹥of cytochrome b562 in the presence of TCEP increased to 45.5 ± 1.9 pN (mean ± SEM), as compared to 27.0 ± 1.4 pN (mean ± SEM) measured without TCEP. We conclude that heme reduction in cytochrome b562 triggered by TCEP increased its mechanical stability by almost 70%. Our present single-molecule level study offers a different perspective on the effect of redox reactions on heme proteins in that that it finds redox-related changes in the mechanical stability of a heme protein. Otherwise, the proposed redox-related change in the molecular flexibility and the folded length of cytochrome b562 could be the basis of protein-based “piezoelectric” nanomaterials and actuators.
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