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.

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 Single-molecule nanopore enzymology

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160230 ◽  
Author(s):  
Kherim Willems ◽  
Veerle Van Meervelt ◽  
Carsten Wloka ◽  
Giovanni Maglia
Keyword(s):  
Membrane Proteins ◽  
Chemical Reactions ◽  
Bias Voltage ◽  
Single Molecule ◽  
Ionic Current ◽  
Enzymatic Reactions ◽  
Current Passing ◽  
Single Molecule Level ◽  
Membrane Pores ◽  
Native Proteins

Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

TGF-β1 Conjugated to Gold Nanoparticles Results in Protein Conformational Changes and Attenuates the Biological Function

Small ◽  
2013 ◽  
Vol 9 (12) ◽  
pp. 2119-2128 ◽  
Author(s):  
Yuh-Shyan Tsai ◽  
Yu-Hung Chen ◽  
Pai-Chiao Cheng ◽  
Hsin-Tzu Tsai ◽  
Ai-Li Shiau ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Conformational Changes ◽  
Biological Function ◽  
Protein Conformational Changes ◽  
Tgf Β1

scholarly journals Single-molecule nanopore sensing of actin dynamics and drug binding

2019 ◽  
Author(s):  
Xiaoyi Wang ◽  
Mark D. Wilkinson ◽  
Xiaoyan Lin ◽  
Ren Ren ◽  
Keith Willison ◽  
...  
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Eukaryotic Cell ◽  
Molecular State ◽  
Drug Binding ◽  
Actin Dynamics ◽  
Label Free ◽  
Single Molecule Level ◽  
Native Proteins ◽  
Voltage Dependent

AbstractActin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.

scholarly journals The relation between intrinsic protein conformational changes and ligand binding

2020 ◽  
Author(s):  
Marijn de Boer
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Conformational Equilibrium ◽  
Biological Function ◽  
Free Protein ◽  
Classical Statistical Mechanics ◽  
Protein Conformational Changes ◽  
Ligand Free ◽  
Non Covalent Interactions ◽  
Covalent Interactions

1ABSTRACTStructural changes in proteins allow them to exist in several conformations. Non-covalent interactions with ligands drive the structural changes, thereby allowing the protein to perform its biological function. Recent findings suggest that many proteins are always in an equilibrium of different conformations and that each of these conformations can be formed by both the ligand-free and ligand-bound protein. By using classical statistical mechanics, we derived the equilibrium probabilities of forming a conformation with and without ligand. We found, under certain conditions, that increasing the probability of forming a conformation by the ligand-free protein also increases the probability of forming the same conformation when the protein has a ligand bound. Further, we found that changes in the conformational equilibrium of the ligand-free protein can increase or decrease the affinity for the ligand.

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 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.

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.

Sign in / Sign up

Export Citation Format

Share Document