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Molecular mechanism of SurA’s chaperoning function to outer membrane proteins revealed by purification-after-crosslinking single-molecule FRET

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Science China Chemistry 2020年第8期63卷 1142-1152页

作者： Chenhui He Sichen Pan Geng Li Xin Sheng Zhao School of Life Sciences Peking UniversityBeijing 100871China Biomedical Pioneering Innovation Center(BIOPIC) Peking UniversityBeijing 100871China Beijing National Laboratory for Molecular Sciences State Key Laboratory for Structural Chemistry of Unstable and Stable Speciesand Department of Chemical BiologyCollege of Chemistry and Molecular EngineeringPeking UniversityBeijing 100871China

SurA is the major chaperone of outer membrane proteins(OMPs)in the periplasm.The molecular mechanism when SurA performs its chaperoning function is still unclear.Here,a purification-after-crosslinking(PAC)procedure was combined with single-molecule fluorescence resonance energy transfer(smfret)to probe the conformations of SurA and OmpC in their complex.We found that SurA in the free state rearranges itself based on the crystal structure,except that the P2 domain moves towards the core domain with two major positions,forming a clamp-like conformation to accommodate OmpC.The obvious rearrangement of the P2 domain of SurA helps SurA to hold OmpC.OmpC attaches to SurA randomly and has the propensity to be near the middle part of the crevice.The noncollapsed and disordered conformations of OMPs provided by the OMPs?SurA complex are important to the subsequent delivery and folding process.

关键词： outer membrane proteins SurA smfret crosslinking

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New insight into the mechanism of DNA polymerase I revealed by single-molecule FRET studies of Klenow fragment

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Chinese Physics B 2022年第8期31卷 705-708页

作者： Rokshana Parvin 贾棋马建兵徐春华陆颖叶方富李明 Beijing National Laboratory for Condensed Matter Physics Institute of PhysicsChinese Academy of SciencesBeijing 100190China School of Physical Sciences University of Chinese Academy of SciencesBeijing 100049China

We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment(exo-)of DNA polymerase I in DNA sequences rich in guanine and cytosine(GC)bases.We find that there exist in the FRET traces numerous ascending jumps,which are induced by the backsliding of Klenow fragment on DNA chains.Our measurements show that the probability of backsliding is closely related to the GC-richness and d NTP concentration:increasing the GC-richness leads to an increase in the backsliding probability,and increasing the d NTP concentration however leads to a decrease in the backsliding probability.These results provide a new insight into the mechanism of DNA polymerase I.

关键词： smfret Klenow fragment GC-richness strand displacement

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Bloom's syndrome protein unfolding G-quadruplexes in two pathways

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Chinese Physics B 2017年第8期26卷 561-564页

作者：赵振业徐春华史婧李菁华马建兵贾棋马东飞李明陆颖 Key Laboratory of Soft Matter Physics.Institute of Physics Chinese Academy of SciencesBeijing 100190China Beijing National Laboratory for Condensed Matter Physics Institute of Physics and School of Physical SciencesUniversity of Chinese Academy of SciencesBeijing 100190China College of Lit'e Science Northwest A&F UniversityXianyang 712100China Material and Energy School Guangdong University of Technology.Guangzhou 510006China

The Bloom helicase （BLM） gene product encodes a DNA helicase that functions in homologous recombination repair to prevent genomic instability. BLM is highly active in binding and unfolding G-quadruplexes （G4）, which are non- canonical DNA structures formed by Hoogsteen base-pairing in guanine-rich sequences. Here we use single-molecule fluorescence resonance energy transfer （smfret） to study the molecular mechanism of BLM-catalysed G4 unfolding and show that BLM unfolds G4 in two pathways. Our data enable us to propose a model in which the HRDC domain functions as a regulator of BLM, depending on the position of the HRDC domain of BLM in action： when HRDC binds to the G4 sequence, BLM may hold G4 in the unfolded state; otherwise, it may remain on the unfolded G4 transiently so that G4 can refold immediately.

关键词： G-quadruplexes BLM helicase smfret

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smfret study of M.HhaI’s conformational changes in pre-catalytic steps

smFRET study of M.HhaI’s conformational changes in pre-cata...

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中国化学会第五届全国生物物理化学会议（NCBPC5）

作者：何姗珊 Xin Sheng Zhao College of Chemistry and Molecular Engineering and Biodynamic Optical Imaging Center （BIOPIC） Peking University

DNA methylation has attracted much attention due to its important role in epigenetic processes.M.HhaI is one of the DNA methyltransferases in bacteria,which catalyzes the methylation of C5 of the inner cytosine in GCGC sequence.Lots of equilibrium and kinetic studies on M.HhaI have been carried out by using ensemble techniques.However,important details could be missed in ensemble studies.We studied the conformational changes of M.HhaI in pre-catalytic steps by using single molecule fluorescence resonance energy transfer（smfret）.The crystal structure shows[1] that upon binding the substrate dsDNA,significant conformational change happens only at the catalytic loop in M.HhaI.By double labeling an AF555-AF647 FRET pair and conducting smfret studies in aqueous condition,we observed more complex behavior thanwhat the crystal structures indicate.The smfret results suggest that there are global conformational changes of M.HhaI before and after binding with DNA.M.HhaI alone undergoes phase transition between the folded and the unfolded states.M.HhaI mainly stays at unfolded state at low salt concentration,and transits to folded state with the increase of salt concentration.By using sm FRET-FCS technique[2],we measured that the phase transition happens at the time scale of several hundred milliseconds,while the catalytic loop motion happens at hundreds of microseconds.These results provide important information about the catalytic property of M.HhaI.

关键词： M.HhaI DNA methyltransferase smfret

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smfret study of interaction between M.HhaI and dsDNA

smFRET study of interaction between M.HhaI and dsDNA

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中国化学会-生物物理化学专业委员会第四届全国生物物理化...

作者：何姗珊北京大学

关键词： smfret M.HhaI

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smfret study of interaction between M.HhaI and dsDNA

smFRET study of interaction between M.HhaI and dsDNA

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第十届全国化学生物学学术会议

作者： Shanshan He Xin Sheng Zhao College of Chemistry and Molecular Engineering and Biodynamic Optical Imaging Center （BIOPIC） Peking University

DNA methylation has attracted much attention due to its important role in epigenetic processes. M.HhaI is one of the DNA methyltransferases in bacteria, which catalyzes the methylation of C5 of the inner cytosine in GCGC sequence. Lots of equilibrium and kinetic studies on M.HhaI have been carried out by using ensemble techniques. However, important details could be missed in ensemble studies. We studied the interaction between dsDNA and M.HhaI by using single molecule fluorescence resonance energy transfer（smfret）. As the crystal structure shows[1], upon binding the substrate dsDNA, significant conformational change happens in M.HhaI—the closure of the catalytic loop. By double labeling an AF555-AF647 FRET pair and conducting smfret studies in aqueous, we observed different results from the crystal structures. The smfret results suggest that there are global conformational changes of M.HhaI before and after binding with DNA, while the crystal structures show only the catalytic loop motion. These results provide important information about the target recognition process of M.HhaI.

关键词： M.HhaI DNA methyltransferase smfret

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OmpC is expansive with the help of chaperone SurA

OmpC is expansive with the help of chaperone SurA

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中国化学会-生物物理化学专业委员会第四届全国生物物理化...

作者：李耕北京大学化学与分子工程学院

The β-barrel outer membrane proteins（OMPs） are located on the outer membrane of Gram-negative bacteria with biological significance. SurA protein has been identified as a periplasmic chaperone that participates in the translocation of OMPs from E. Coli’s inner membrane to outer membrane[1]. Using single molecule fluorescence resonance energy transfer（smfret）, we studied the interaction between outer membrane protein C（OmpC） and SurA with/without the presence of urea. We found that just like in urea denaturing condition, OmpC that binds to SurA showed an expansive conformation. This phenomenon may be important in understanding the mechanism of the biogenesis of OMPs.

关键词： OmpC SurA smfret expansive conformation

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Dispersed OmpT may be partially folded

Dispersed OmpT may be partially folded

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中国化学会-生物物理化学专业委员会第四届全国生物物理化...

作者：卜佩璇赵新生 College of Chemistry and Molecular Engineering and Biodynamic Optical Imaging Center（BIOPIC） Peking University

It is believed that outer membrane proteins in Gram-negative bacteria are protected by chaporones during their translocation through the periplasm because they are structureless and prone to aggregation. The previous work on OmpC supported such an opinion. However, recently we found that OmpT has less tendency to aggregate and may be partially folded in its dispersed form. By using single molecule fluorescence resonance energy transfer（smfret）, the equilibrium denaturation of OmpT induced by urea has been investigated. Double-cysteine mutant（S1C/F297C） of OmpT is engineered and labeled with FRET dyes. We observed subpopulations of denatured and partially folded form of OmpT and the two subpopulations show two-state transition as urea concentration changes. These resluts may help better understanding of outer membrane protein folding.

关键词： OmpT smfret urea denaturation partial folding

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Single–molecule study on the biogenesis of OMPs

Single–molecule study on the biogenesis of OMPs

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第十届全国化学生物学学术会议

作者： Xin Sheng Zhao Department of Chemical Biology College of Chemistry Peking University

The biogenesis of beta-barrel outer membrane proteins（OMPs） in Gram–negative bacteria is a complex process. The transportation of OMPs through the periplasm is a key step during the process. The periplasm is an aqueous and oxidizing environment,so the insoluble and aggregation–prone OMPs must be kept in a folding competent state during their traverse in the periplasm. SurA, Skp and DegP are the most prominent quality control factors to protect and guide OMPs across the periplasm.Both SurA and Skp act as chaperones, and they are considered to configure a partially redundant network with SurA playing the major role and Skp the minor. By using single–molecule fluorescence resonance energy transfer（smfret） and fluorescence correlation spectroscopy（FCS）, we investigated the molecular mechanism about how these quality control factors work together. Our study reveals the functional distinctions between Skp and SurA. Especially, Skp is not redundant and will be more significant under stressed conditions. Based on our study and literatures a model for the OMPs transportation in the periplasm has been proposed.

关键词： smfret FCS OMPs biogenesis

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Mechanism on protection of E.coli outer membrane proteins by periplasmic chaperones SurA and Skp

Mechanism on protection of E.coli outer membrane proteins by...

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第十届全国化学生物学学术会议

作者： Geng Li Xin Sheng Zhao Beijing National Laboratory for Molecular Sciences State Key Laboratory for Structural Chemistry of Unstable and Stable SpeciesDepartment of Chemical BiologyCollege of Chemistry and Molecular Engineeringand Biodynamic Optical Imaging Center （BIOPIC）Peking University

SurA and Skp are both periplasmic chaperones in the biogenesis of E. coli β–barrel outer membrane proteins（OMPs）. It is commonly assumed that SurA plays a major role, whereas Skp is a minor factor. However, there is no molecular evidence whether their roles are redundant or not. By using different dilution methods, we obtained monodispersed and aggregated OMPs and studied their interactions with Skp and SurA by single–molecule fluorescence resonance energy transfer（smfret） and fluorescence correlation spectroscopy（FCS）. We found that Skp has the ability to dissolve the aggregated OMPs, while SurA can hardly convert the aggregated OMPs into a monodispersed form, and OMPs protected by the two chaperones possess different conformations and stoichiometries. Our study demonstrates the functional distinctions between Skp and SurA. Especially, Skp is not redundant and will be more significant under stressed conditions. On the other hand, by using FRET technique, we studied the molecular interaction between SurA and outer membrane protein C（OmpC） from conformations to kinetics. Our results show that after binding to OmpC, SurA basically maintains its structure but somewhat expanded, in which the distance between the N and P1 domains is most stretched, and OmpC behaves like an extended random coil in the SurA-OmpC complex. Based on our conformational and kinetic information and previous works, we propose that OMPs anchors on the crevice in SurA’s core module first, then is wrapped around SurA, and finally one of its terminals settles down on the P2 domain.This manner of interaction may be essential for SurA to convey OMPs to BAM system for the folding and assembling.

关键词： Outer membrane proteins Chaperone smfret FCS

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