scholarly journals Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

2016 ◽  
Vol 193 ◽  
pp. 471-485 ◽  
Author(s):  
Robert P. Johnson ◽  
Rukshan T. Perera ◽  
Aaron M. Fleming ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Dna Duplexes ◽  
Base Flipping ◽  
Single Molecule Level ◽  
Dna Mismatch ◽  
Double Stranded Dna ◽  
Measured Activation Energy ◽  
Measured Activation ◽  
Significant Mechanism

Unique, two-state modulating current signatures are observed when a cytosine–cytosine mismatch pair is confined at the 2.4 nm latch constriction of the α-hemolysin (αHL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180°. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside αHL are used to derive thermodynamic (ΔH, ΔS) and kinetic (EA) parameters for base flipping of a cytosine at an unstable cytosine–cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of αHL out of the helix (18 ± 1 kcal mol−1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the αHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.

scholarly journals Characterization of Fluorescent Base Analogs to Study DNA Base Flipping at the Single Molecule Level

2013 ◽  
Vol 104 (2) ◽  
pp. 346a
Author(s):  
Xochitl A. Sosa-Vazquez ◽  
Matthew Vander-Schuur ◽  
Liza Valencia ◽  
Elvin A. Aleman
Keyword(s):  
Single Molecule ◽  
Base Flipping ◽  
Single Molecule Level ◽  
Dna Base

scholarly journals Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy

2019 ◽  
Vol 116 (23) ◽  
pp. 11247-11252 ◽  
Author(s):  
Toru Kondo ◽  
Jesse B. Gordon ◽  
Alberta Pinnola ◽  
Luca Dall’Osto ◽  
Roberto Bassi ◽  
...  
Keyword(s):  
Correlation Function ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Light Harvesting ◽  
Building Blocks ◽  
Complex Stress ◽  
Correlation Spectroscopy ◽  
Light Harvesting Complex ◽  
Single Molecule Level

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.

scholarly journals Optical imaging of single protein size, charge, mobility, binding and conformational change

2018 ◽  
Author(s):  
Guanzhong Ma ◽  
Hao Zhu ◽  
Zijian Wan ◽  
Yunze Yang ◽  
Shaopeng Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Near Field ◽  
Protein Analysis ◽  
Imaging Method ◽  
Charge Mobility ◽  
Single Molecule Level ◽  
Protein Size ◽  
Flexible Polymer ◽  
Protein Oscillation

AbstractProtein analysis has relied on electrophoresis, mass spectroscopy and immunoassay, which separate, detect and identify proteins based on the size, charge, mobility and binding to antibodies. However, measuring these quantities at the single molecule level has not been possible. We tether a protein to a surface with a flexible polymer, drive the protein into mechanical oscillation with an alternating electric field, and image the protein oscillation with a near field imaging method, from which we determine the size, charge, mobility of the protein. We also measure binding of antibodies to single proteins and ligand binding-induced conformational changes in single proteins. This work provides new capabilities for protein analysis and disease biomarker detection at the single molecule level.

scholarly journals γ-Hemolysin Nanopore Is Sensitive to Guanine-to-Inosine Substitutions in Double-Stranded DNA at the Single-Molecule Level

2018 ◽  
Vol 140 (43) ◽  
pp. 14224-14234 ◽  
Author(s):  
Cherie S. Tan ◽  
Aaron M. Fleming ◽  
Hang Ren ◽  
Cynthia J. Burrows ◽  
Henry S. White
Keyword(s):  
Single Molecule ◽  
Single Molecule Level ◽  
Double Stranded Dna

scholarly journals Structural titration of receptor ion channel GLIC gating by HS-AFM

2018 ◽  
Vol 115 (41) ◽  
pp. 10333-10338 ◽  
Author(s):  
Yi Ruan ◽  
Kevin Kao ◽  
Solène Lefebvre ◽  
Arin Marchesi ◽  
Pierre-Jean Corringer ◽  
...  
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
High Speed ◽  
Conformational Dynamics ◽  
Protein Protein Interactions ◽  
Ion Conductance ◽  
Single Molecule Level ◽  
Selective Channel

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein–protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

DNA polymerase activity at the single-molecule level

2011 ◽  
Vol 39 (2) ◽  
pp. 595-599 ◽  
Author(s):  
Joshua P. Gill ◽  
Jun Wang ◽  
David P. Millar
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Complexes ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Single Molecule Level ◽  
Nucleotide Incorporation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Conformational Rearrangements ◽  
Dna Complex

DNA polymerases are essential enzymes responsible for replication and repair of DNA in all organisms. To replicate DNA with high fidelity, DNA polymerases must select the correct incoming nucleotide substrate during each cycle of nucleotide incorporation, in accordance with the templating base. When an incorrect nucleotide is sometimes inserted, the polymerase uses a separate 3′→5′ exonuclease to remove the misincorporated base (proofreading). Large conformational rearrangements of the polymerase–DNA complex occur during both the nucleotide incorporation and proofreading steps. Single-molecule fluorescence spectroscopy provides a unique tool for observation of these dynamic conformational changes in real-time, without the need to synchronize a population of DNA–protein complexes.

scholarly journals MutL traps MutS at a DNA mismatch

2015 ◽  
Vol 112 (35) ◽  
pp. 10914-10919 ◽  
Author(s):  
Ruoyi Qiu ◽  
Miho Sakato ◽  
Elizabeth J. Sacho ◽  
Hunter Wilkins ◽  
Xingdong Zhang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Thermus Aquaticus ◽  
Dna Mismatch ◽  
Sliding Clamp ◽  
Single Molecule Fret ◽  
Mismatch Detection ◽  
Current Thinking ◽  
Processivity Factor ◽  
Subsequent Activation

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

Minimalist Approaches to Protein Labelling: Getting the Most Fluorescent Bang for Your Steric Buck

2014 ◽  
Vol 67 (5) ◽  
pp. 686 ◽  
Author(s):  
Lee C. Speight ◽  
Moumita Samanta ◽  
E. James Petersson
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Unnatural Amino Acid ◽  
Spectroscopic Techniques ◽  
Environmental Sensitivity ◽  
Protein Labelling ◽  
Single Molecule Level ◽  
Wavelength Absorption

Fluorescence methods allow one to monitor protein conformational changes, protein–protein associations, and proteolysis in real time, at the single molecule level and in living cells. The information gained in such experiments is a function of the spectroscopic techniques used and the strategic placement of fluorophore labels within the protein structure. There is often a trade-off between size and utility for fluorophores, whereby large size can be disruptive to the protein’s fold or function, but valuable characteristics, such as visible wavelength absorption and emission or brightness, require sizable chromophores. Three major types of fluorophore readouts are commonly used: (1) Förster resonance energy transfer (FRET); (2) photoinduced electron transfer (PET); and (3) environmental sensitivity. This review focuses on those probes small enough to be incorporated into proteins during ribosomal translation, which allows the probes to be placed on the interiors of proteins as they are folded during synthesis. The most broadly useful method for doing so is site-specific unnatural amino acid (UAA) mutagenesis. We discuss the use of UAA probes in applications relying on FRET, PET, and environmental sensitivity. We also briefly review other methods of protein labelling and compare their relative merits to UAA mutagenesis. Finally, we discuss small probes that have thus far been used only in synthetic peptides, but which have unusual value and may be candidates for incorporation using UAA methods.

scholarly journals Conformation switching of single native proteins revealed by nanomechanical probing without a pulling force

Nanoscale ◽  
2019 ◽  
Vol 11 (42) ◽  
pp. 19933-19942
Author(s):  
Fabiola A. Gutiérrez-Mejía ◽  
Christian P. Moerland ◽  
Leo J. van IJzendoorn ◽  
Menno W. J. Prins
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Biological Function ◽  
Single Molecule Level ◽  
Native Proteins ◽  
Protein Conformational Changes ◽  
P Protein ◽  
Heterogeneous Nature ◽  
Pulling Force

Protein conformational changes are essential to biological function, and the heterogeneous nature of the corresponding protein states provokes an interest to measure conformational changes at the single molecule level.

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