scholarly journals A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers

2018 ◽  
Vol 150 (2) ◽  
pp. 355-365 ◽  
Author(s):  
Rahul Chadda ◽  
Lucy Cliff ◽  
Marley Brimberry ◽  
Janice L. Robertson
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Equilibrium Constants ◽  
Correction Method ◽  
Equilibrium Studies ◽  
Model Free ◽  
Liposome Size ◽  
New Type ◽  
Protein Density

The thermodynamic reasons why membrane proteins form stable complexes inside the hydrophobic lipid bilayer remain poorly understood. This is largely because of a lack of membrane–protein systems amenable for equilibrium studies and a limited number of methods for measuring these reactions. Recently, we reported the equilibrium dimerization of the CLC-ec1 Cl−/H+ transporter in lipid bilayers (Chadda et al. 2016. eLife. https://doi.org/10.7554/eLife.17438), which provided a new type of model system for studying protein association in membranes. The measurement was conducted using the subunit-capture approach, involving passive dilution of the protein in large multilamellar vesicles, followed by single-molecule photobleaching analysis of the Poisson distribution describing protein encapsulation into extruded liposomes. To estimate the fraction of dimers (FDimer) as a function of protein density, the photobleaching distributions for the nonreactive, ideal monomer and dimer species must be known so that random co-capture probabilities can be accounted for. Previously, this was done by simulating the Poisson process of protein reconstitution into a known size distribution of liposomes composed of Escherichia coli polar lipids (EPLs). In the present study, we investigate the dependency of FDimer and ΔG° on the modeling through a comparison of different liposome size distributions (EPL versus 2:1 POPE/POPG). The results show that the estimated FDimer values are comparable, except at higher densities when liposomes become saturated with protein. We then develop empirical controls to directly measure the photobleaching distributions of the nonreactive monomer (CLC-ec1 I201W/I422W) and ideal dimer (WT CLC-ec1 cross-linked by glutaraldehyde or CLC-ec1 R230C/L249C cross-linked by a disulfide bond). The measured equilibrium constants do not depend on the correction method used, indicating the robustness of the subunit-capture approach. This strategy therefore presents a model-free way to quantify protein dimerization in lipid bilayers, offering a simplified strategy in the ongoing effort to characterize equilibrium membrane–protein reactions in membranes.

scholarly journals Occupancy distributions of membrane proteins in heterogeneous liposome populations

2019 ◽  
Author(s):  
Lucy Cliff ◽  
Rahul Chadda ◽  
Janice L. Robertson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Skewed Distribution ◽  
Liposome Size ◽  
Protein Incorporation ◽  
Size Heterogeneity ◽  
The Right ◽  
And Function

AbstractMeasurements of membrane protein structure and function often rely on reconstituting the protein into lipid bilayers through the formation of liposomes. Many measurements conducted in proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, require some understanding of the occupancy statistics of the liposome population for correct interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein incorporation can be described by the Poisson distribution. However, in reality, liposomes are heterogeneous, which alters the statistics of occupancy in several ways. Here, we determine the liposome occupancy distribution for membrane protein reconstitution while taking into account liposome size heterogeneity. We calculate the protein occupancy for a homogenous population of liposomes with radius r = 200 nm, representing an idealization of vesicles extruded through 400 nm pores and compare it to the right-skewed distribution of 400 nm 2:1 POPE:POPG vesicles. As is the case for E. coli polar lipids, this synthetic composition yields a sub-population of small liposomes, ∼25 nm in radius with a long tail of larger vesicles. Previously published microscopy data of the co-localization of the CLC-ec1 Cl-/H+ transporter with liposomes, and vesicle occupancy measurements using functional transport assays, shows agreement with the heterogeneous 2:1 POPE:POPG population. Next, distributions of 100 nm and 30 nm extruded 2:1 POPE:POPG liposomes are measured by cryo-electron microscopy, demonstrating that extrusion through smaller pores does not shift the peak, but reduces polydispersity arising from large liposomes. Single-molecule photobleaching analysis of CLC-ec1-Cy5 shows the 30 nm extruded population increases the ‘Poisson-dilution’ range, reducing the probability of vesicles with more than one protein at higher protein/lipid densities. These results demonstrate that the occupancy distributions of membrane proteins into vesicles can be accurately predicted in heterogeneous populations with experimental knowledge of the liposome size distribution.

scholarly journals The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Rahul Chadda ◽  
Venkatramanan Krishnamani ◽  
Kacey Mersch ◽  
Jason Wong ◽  
Marley Brimberry ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Protein Complexes ◽  
New Approach ◽  
Membrane Protein Folding ◽  
Protein Interfaces ◽  
New Type ◽  
Membrane Protein Complexes ◽  
Physical Forces

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

scholarly journals A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers

2017 ◽  
Author(s):  
Rahul Chadda ◽  
Lucy Cliff ◽  
Marley Brimberry ◽  
Janice L. Robertson
Keyword(s):  
Size Distribution ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Probability Distributions ◽  
E Coli ◽  
Model Free ◽  
Wide Range ◽  
Alternate Model ◽  
Liposome Size ◽  
Dimerization Reaction

ABSTRACTWe previously reported the equilibrium dimerization reaction of the CLC-ec1 Cl-/H+ transporter in 2:1 POPE/POPG membranes (Chadda et al. 2016). This was determined by measuring the probability distributions of subunit capture into extruded liposomes by single-molecule photobleaching analysis across a wide range of subunit/lipid mole fraction densities. In this approach, knowledge of the liposome size distribution is necessary in order to correct the data for random co-capture events and extract the underlying dimerization reaction. For this we used a previously reported cryo-electron microscopy (cryo-EM) measured size distribution of 400 nm extruded liposomes made of E. coli polar lipids (Walden et al. 2007). While the model and data agreed at low densities, we observed systematic inaccuracies at higher densities limiting our ability to extract FDimer in this range. To address this issue, we measured the 400 nm extruded 2:1 POPE/POPG liposome size distribution by cryo-EM and found that there is a small, but significant amount of larger liposomes in the population. Re-analysis of the I201W/I422W ‘WW’ photobleaching data using this distribution shows that the protein is monomeric in the membrane and can serve as an experimental control. Dimer controls were constructed by glutaraldehyde cross-linking of C85A/H234C ‘WT’ or introducing R230C/L249C, which forms a spontaneous disulfide bond. Determination of FDimer based on the experimental controls yields improved fits and no change in the previously reported ΔG° values, providing an alternate model-free approach to measuring CLC-ec1 dimerization in membranes.

scholarly journals Aggregation-related quenching of LHCII in liposomes revealed by single-molecule spectroscopy

2020 ◽  
Author(s):  
Marijonas Tutkus ◽  
Jevgenij Chmeliov ◽  
Gediminas Trinkunas ◽  
Parveen Akhtar ◽  
Petar H. Lambrev ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Lipid Membranes ◽  
Photochemical Quenching ◽  
Liposome Size ◽  
Function Relationship ◽  
Structure And Function Relationship ◽  
Protein Density ◽  
And Function

AbstractIncorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

Self-assembling Peptide Detergent A6D Directly Associates with Bovine Rhodopsin and Forms Lipid-like Vesicles

2004 ◽  
Vol 845 ◽  
Author(s):  
Xiaojun Zhao ◽  
Yusuke Nagai ◽  
Shuguang Zhang
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Self Assembly ◽  
Protein Structures ◽  
Detergent Solution ◽  
Self Assembling ◽  
Bovine Rhodopsin ◽  
Hydrophobic Tail ◽  
New Type

ABSTRACTMembrane protein study critically depends on detergents, which are amphilhilic molecules containing a hydrophilic “head” and a hydrophobic “tail” to mimic biological lipid bilayers to stabilize membrane proteins. However, detergents are not fully equivalent to lipid bilayers and in fact they only partly mimic lipid bilayers function. Consequently, membrane proteins in detergent solution are more or less denatured because detergents can not effectively stabilize membrane protein structures. Therefore, it is urgent to develop new types of detergents for more effectively stabilizing membrane proteins. Previously, we have reported a new type of self-assembly peptide detergents containing a hydrophilic head composed of either a negatively charged aspartic acid or a positively charged lysine and a tail of hydrophobic amino acids of six connective alanines. This new peptide detergent has been shown to be more effective for protecting membrane protein PS I structure than that the conventional detergent does. However, what type of physical structures peptide detergent can form is unclear yet. Here we presented our AFM and DSL analysis of the peptide detergent A6D, which not only form mixed micelles with n-Octyl-beta-D-Glucoside (OG) to solubilize membrane protein rhodopsin, but also can mimic lipid bilayers to keep rhodopsin in lipid-like vesicles for its structure preservation.

Single Molecule Height Measurements on a Membrane Protein in Nanometer-Scale Phospholipid Bilayer Disks

Langmuir ◽  
2000 ◽  
Vol 16 (14) ◽  
pp. 5993-5997 ◽  
Author(s):  
Timothy H. Bayburt ◽  
Joseph W. Carlson ◽  
Stephen G. Sligar
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Phospholipid Bilayer ◽  
Nanometer Scale

scholarly journals Applications of Single-Molecule Methods to Membrane Protein Folding Studies

2018 ◽  
Vol 430 (4) ◽  
pp. 424-437 ◽  
Author(s):  
Robert E. Jefferson ◽  
Duyoung Min ◽  
Karolina Corin ◽  
Jing Yang Wang ◽  
James U. Bowie
Keyword(s):  
Protein Folding ◽  
Membrane Protein ◽  
Single Molecule ◽  
Membrane Protein Folding

scholarly journals The association behaviour of β-lactamases. Sedimentation equilibrium studies in ammonium sulphate solutions

1986 ◽  
Vol 237 (2) ◽  
pp. 511-517 ◽  
Author(s):  
E H Braswell ◽  
J R Knox ◽  
J M Frère
Keyword(s):  
Equilibrium Constants ◽  
Solubility Limit ◽  
Sedimentation Equilibrium ◽  
Association Model ◽  
Sodium Cacodylate ◽  
Dimerization Constant ◽  
Equilibrium Studies ◽  
Crystal Forms ◽  
Beta Lactamases ◽  
Equilibrium Sedimentation

The beta-lactamases (EC 3.5.2.6) from TEM plasmid RP4, Bacillus licheniformis 749/C and Enterobacter cloacae P99 were studied in solution over a wide concentration range by equilibrium sedimentation. Though crystal symmetries indicate that all three enzymes are potentially dimeric in their crystal forms, in 50 mM-sodium cacodylate at pH 6.5 the enzymes show only a small tendency to associate, indicated by a weight-average Mr (Mw) at 3% (w/v) concentration about 9% greater than that of the monomer. Although the mode of association could not be determined, this extent of association corresponded to a dimerization constant of about 2 × 10(2) M-1. In 2.1 M-(NH4)2SO4 the B. licheniformis enzyme shows some association at concentrations over 1%, displaying an Mw value at 7% concentration about 60% more than the monomer. Under the same conditions Mw for the Entero. P99 enzyme is about 60% greater than the monomer near the solubility limit of about 2%. However, the Mw for the TEM enzyme is over twice that of the monomer at its solubility limit (3%) in 1.7 M-(NH4)2SO4. Fitting the sedimentation data of the TEM enzyme in 1.7 M-(NH4)2SO4 with a dimerization model and an indefinite-isodesmic-association model yielded equilibrium constants of 1.5 × 10(4) and 3.3 × 10(2) M-1 respectively, with the indefinite-isodesmic model giving the better fit. Fitting the data for the other two enzymes yielded values of 1.4 × 10(3) and 1.7 × 10(2) M-1 respectively for the Entero. P99 enzyme and 4.5 × 10(2) and 45 M-1 respectively for the B. licheniformis enzyme. It could not be determined which model was the better fit for these two enzymes. Since none of the beta-lactamases studied here showed strong evidence of the terminal aggregate being a dimer, we conclude that crystalline dimers, if they exist, will not be tightly associated or physiologically significant.

scholarly journals EXPRESS: Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

2021 ◽  
pp. 000370282110099
Author(s):  
Ziyu Yang ◽  
Haiqi Xu ◽  
Jiayu Wang ◽  
Wei Chen ◽  
Meiping Zhao
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Membrane Receptors ◽  
Biological Macromolecules ◽  
Dynamics Analysis ◽  
Single Molecule Fluorescence ◽  
Membrane Protein Dynamics

Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

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