The ribosome,a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter,coordinates numerous ligands,factors,and regulators to translate proteins with high fidelity and speed.Understanding its complex func...The ribosome,a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter,coordinates numerous ligands,factors,and regulators to translate proteins with high fidelity and speed.Understanding its complex functions necessitates multiperspective observations.We developed a dualFRET single-molecule Förste Resonance Energy Transfer method(dual-smFRET),allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G(EF-G)conformations in the same complex,in a 10 s time window.By synchronizing laser shutters and motorized filter sets,two FRET signals are captured in consecutive 5 s intervals with a time gap of 50-100 ms.We observed distinct fluorescent emissions from single-,double-,and quadruple-labeled ribosome complexes.Through comprehensive spectrum analysis and correction,we distinguish and correlate conformational changes in two parts of the ribosome,offering additional perspectives on its coordination and timing during translocation.Our setup’s versatility,accommodating up to six FRET pairs,suggests broader applications in studying large biomolecules and various biological systems.展开更多
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）, whi...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.展开更多
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 ...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.展开更多
The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses(CoV),particularly the highly conserved nonstructural CoV proteins(nsp).Among these,the nsp13 helicas...The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses(CoV),particularly the highly conserved nonstructural CoV proteins(nsp).Among these,the nsp13 helicase of severe pathogenic MERS-CoV,SARS-CoV-2,and SARS-CoV is one of the most preserved CoV nsp.Utilizing single-molecule FRET,we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed.If a different nucleotide is introduced,these steps diminish to 3−4 bp.Dwell-time analysis revealed 3−4 concealed steps within each unwinding process,which suggests the hydrolysis of 3−4 dTTP.Combining our observations with previous studies,we propose an unwinding model of CoV nsp13 helicase.This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA,even as the helicase core domain has advanced over 3−4 bp,thereby inducing accumulated strain on the nsp13-DNA complex.Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.展开更多
Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular dr...Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that controlbiomolecular systems.Single-molecule fluorescence resonance energy transfer(smFRET)is a powerful technique to observe inreal-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states.Currently,this technique is fundamentally limited by irreversible photobleaching,causing the untimely end of the experiment andthus,a narrow temporal bandwidth of≤3 orders of magnitude.Here,we introduce“DyeCycling”,a measurement scheme withwhich we aim to break the photobleaching limit in smFRET.We introduce the concept of spontaneous dye replacement bysimulations,and as an experimental proof-of-concept,we demonstrate the intermittent observation of a single biomolecule forone hour with a time resolution of milliseconds.Theoretically,DyeCycling can provide>100-fold more information per singlemolecule than conventional smFRET.We discuss the experimental implementation of DyeCycling,its current and fundamentallimitations,and specific biological use cases.Given its general simplicity and versatility,DyeCycling has the potential torevolutionize the field of time-resolved smFRET,where it may serve to unravel a wealth of biomolecular dynamics by bridgingfrom milliseconds to the hour range.展开更多
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 wa...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.展开更多
基金supported by NIGMS grant:R01GM111452,NSF:2130427the Welch Foundation:E-1721 to Y.Wang。
文摘The ribosome,a 2.6 megadalton biomolecule measuring approximately 20 nm in diameter,coordinates numerous ligands,factors,and regulators to translate proteins with high fidelity and speed.Understanding its complex functions necessitates multiperspective observations.We developed a dualFRET single-molecule Förste Resonance Energy Transfer method(dual-smFRET),allowing simultaneous observation and correlation of tRNA dynamics and Elongation Factor G(EF-G)conformations in the same complex,in a 10 s time window.By synchronizing laser shutters and motorized filter sets,two FRET signals are captured in consecutive 5 s intervals with a time gap of 50-100 ms.We observed distinct fluorescent emissions from single-,double-,and quadruple-labeled ribosome complexes.Through comprehensive spectrum analysis and correction,we distinguish and correlate conformational changes in two parts of the ribosome,offering additional perspectives on its coordination and timing during translocation.Our setup’s versatility,accommodating up to six FRET pairs,suggests broader applications in studying large biomolecules and various biological systems.
基金supported by the National Natural Science Foundation of China(Grant Nos.11674382,11574381,and 11574382)the Key Research Program of Frontier Sciences,Chinese Academy of Sciences(Grant No.QYZDJ-SSW-SYS014)
文摘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.
基金Project supported by the National Natural Science Foundation of China(Grant No.12090051)the CAS Key Research Program of Frontier Sciences(Grant Nos.QYZDJSSW-SYS014 and ZDBS-LY-SLH015)the Youth Innovation Promotion Association of CAS(Grant No.2017015)。
文摘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.
基金supported by CRP-ICGEB Research Grant 2019(Grant number:CRP/CHN19-02)National Key Research and Development Program of China(Grant number:2016YFD0500300)supported by the Special Coronavirus(COVID-19)Research Pilot Grant Program from University of Cincinnati College of Medicine.
文摘The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses(CoV),particularly the highly conserved nonstructural CoV proteins(nsp).Among these,the nsp13 helicase of severe pathogenic MERS-CoV,SARS-CoV-2,and SARS-CoV is one of the most preserved CoV nsp.Utilizing single-molecule FRET,we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed.If a different nucleotide is introduced,these steps diminish to 3−4 bp.Dwell-time analysis revealed 3−4 concealed steps within each unwinding process,which suggests the hydrolysis of 3−4 dTTP.Combining our observations with previous studies,we propose an unwinding model of CoV nsp13 helicase.This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA,even as the helicase core domain has advanced over 3−4 bp,thereby inducing accumulated strain on the nsp13-DNA complex.Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.
文摘Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that controlbiomolecular systems.Single-molecule fluorescence resonance energy transfer(smFRET)is a powerful technique to observe inreal-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states.Currently,this technique is fundamentally limited by irreversible photobleaching,causing the untimely end of the experiment andthus,a narrow temporal bandwidth of≤3 orders of magnitude.Here,we introduce“DyeCycling”,a measurement scheme withwhich we aim to break the photobleaching limit in smFRET.We introduce the concept of spontaneous dye replacement bysimulations,and as an experimental proof-of-concept,we demonstrate the intermittent observation of a single biomolecule forone hour with a time resolution of milliseconds.Theoretically,DyeCycling can provide>100-fold more information per singlemolecule than conventional smFRET.We discuss the experimental implementation of DyeCycling,its current and fundamentallimitations,and specific biological use cases.Given its general simplicity and versatility,DyeCycling has the potential torevolutionize the field of time-resolved smFRET,where it may serve to unravel a wealth of biomolecular dynamics by bridgingfrom milliseconds to the hour range.
基金supported by the National Natural Science Foundation of China(21233002,21521003)the National Key Basic Research Special Foundation of China(2012CB917304)。
文摘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.
