Mechanical Unfolding and Refolding of NanoLuc via Single- Molecule Force Spectroscopy and Computer Simulations

被引:2
作者
Apostolidou, Dimitra [2 ]
Zhang, Pan [1 ]
Yang, Weitao [1 ]
Marszalek, Piotr E. [2 ]
机构
[1] Duke Univ, Dept Chem, Durham, NC 27708 USA
[2] Duke Univ, Dept Mech Engn & Mat Sci, Durham, NC 27708 USA
基金
美国国家科学基金会;
关键词
DEEP-SEA SHRIMP; OPLOPHORUS LUCIFERASE; PROTEIN; FLUORESCENCE; PATHWAY; COMPONENT; DYNAMICS; REPORTER; REVEALS;
D O I
10.1021/acs.biomac.2c00997
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912- Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912 construct. Our SMFS results confirm that Nluc's unfolding proceeds similarly in all constructs; however, Nluc's refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.
引用
收藏
页码:5164 / 5178
页数:15
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