Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Structural basis of pH-dependent activation in a CLC transporter.

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl- pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers.

The CLC-ec1 chloride/proton antiporter is a membrane-embedded homodimer with subunits that can dissociate and associate, but the thermodynamic driving forces favor the assembled dimer at biological densities. Yet, the physical reasons for this stability are confounding as dimerization occurs via the burial of hydrophobic interfaces away from the lipid solvent. For binding of nonpolar surfaces in aqueous solution, the driving force is often attributed to the hydrophobic effect, but this should not apply in the membrane since there is very little water. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG°. To ensure that the reaction reached equilibrium at different temperatures, we utilized a Förster resonance energy transfer assay to report on relaxation kinetics of subunit exchange as a function of temperature. Equilibration times were then applied to measure CLC-ec1 dimerization isotherms at different temperatures using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in Escherichia coli-like membranes exhibits a nonlinear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects such as the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the nonbilayer defect required to solvate the monomeric state is one source of the observed change in heat capacity and indicates the existence of a generalizable driving force for protein association in membranes.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

Mechanistic insight into AP-endonuclease 1 cleavage of abasic sites at stalled replication fork mimics.

Many types of damage, including abasic sites, block replicative DNA polymerases causing replication fork uncoupling and generating ssDNA. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at replication forks remain elusive. Here, we investigate APE1 cleavage of abasic substrates that mimic APE1 interactions at stalled replication forks or gaps. We determine that APE1 has robust activity on these substrates, like dsDNA, and report rates for cleavage and product release. X-ray structures visualize the APE1 active site, highlighting an analogous mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

An Aurora B-RPA signaling axis secures chromosome segregation fidelity.

Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers.

The CLC-ec1 chloride/proton antiporter is a membrane embedded homodimer where subunits can dissociate and associate, but the thermodynamic driving forces favor the assembled form at biological densities. Yet, the physical reasons for this stability are confounding since binding occurs via the burial of hydrophobic protein interfaces yet the hydrophobic effect should not apply since there is little water within the membrane. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG° . To ensure that the reaction reached equilibrium under changing conditions, we utilized a Förster Resonance Energy Transfer based assay to report on the relaxation kinetics of subunit exchange as a function of temperature. These equilibration times were then applied to measure CLC-ec1 dimerization isotherms as a function of temperature using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in E. coli membranes exhibits a non-linear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects including the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the non-bilayer defect required to solvate the monomeric state is the molecular source of this large change in heat capacity and is a major and generalizable driving force for protein association in membranes.

Homodecameric Rad52 promotes single-position Rad51 nucleation in homologous recombination.

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

Rtt105 regulates RPA function by configurationally stapling the flexible domains.

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states.

Over two-thirds of integral membrane proteins of known structure assemble into oligomers. Yet, the forces that drive the association of these proteins remain to be delineated, as the lipid bilayer is a solvent environment that is both structurally and chemically complex. In this study, we reveal how the lipid solvent defines the dimerization equilibrium of the CLC-ec1 Cl-/H+ antiporter. Integrating experimental and computational approaches, we show that monomers associate to avoid a thinned-membrane defect formed by hydrophobic mismatch at their exposed dimerization interfaces. In this defect, lipids are strongly tilted and less densely packed than in the bulk, with a larger degree of entanglement between opposing leaflets and greater water penetration into the bilayer interior. Dimerization restores the membrane to a near-native state and therefore, appears to be driven by the larger free-energy cost of lipid solvation of the dissociated protomers. Supporting this theory, we demonstrate that addition of short-chain lipids strongly shifts the dimerization equilibrium toward the monomeric state, and show that the cause of this effect is that these lipids preferentially solvate the defect. Importantly, we show that this shift requires only minimal quantities of short-chain lipids, with no measurable impact on either the macroscopic physical state of the membrane or the protein's biological function. Based on these observations, we posit that free-energy differentials for local lipid solvation define membrane-protein association equilibria. With this, we argue that preferential lipid solvation is a plausible cellular mechanism for lipid regulation of oligomerization processes, as it can occur at low concentrations and does not require global changes in membrane properties.

A cell's outer membrane is made of molecules called lipids, which band together to form a flexible thin film, just two molecules thick. This membrane is dotted with proteins that transport materials in to and out of cells. Most of these membrane proteins join with other proteins to form structures known as oligomers. Except, how membrane-bound proteins assemble into oligomers ­ the physical forces driving these molecules to take shape ­ remains unclear. This is partly because the structural, physical and chemical properties of fat-like lipid membranes are radically different to the cell's watery interior. Consequently, the conditions under which membrane oligomers form are distinct from those surrounding proteins inside cells. Membrane proteins are also more difficult to study and characterize than water-soluble proteins inside the cell, and yet many therapeutic drugs such as antibiotics specifically target membrane proteins. Overall, our understanding of how the unique properties of lipid membranes affect the formation of protein structures embedded within, is lacking and warrants further investigation. Now, Chadda, Bernhardt et al. focused on one membrane protein, known as CLC, which tends to exist in pairs ­ or dimers. To understand why these proteins form dimers (a process called dimerization) Chadda, Bernhardt et al. first used computer simulations, and then validated the findings in experimental tests. These complementary approaches demonstrated that the main reason CLC proteins 'dimerize' lies in their interaction with the lipid membrane, and not the attraction of one protein to its partner. When CLC proteins are on their own, they deform the surrounding membrane and create structural defects that put the membrane under strain. But when two CLC proteins join as a dimer, this membrane strain disappears ­ making dimerization the more stable and energetically favorable option. Chadda, Bernhardt et al. also showed that with the addition of a few certain lipids, specifically smaller lipids, cell membranes become more tolerant of protein-induced structural changes. This might explain how cells could use various lipids to fine-tune the activity of membrane proteins by controlling how oligomers form. However, the theory needs to be examined further. Altogether, this work has provided fundamental insights into the physical forces shaping membrane-bound proteins, relevant to researchers studying cell biology and pharmacology alike.

Occupancy distributions of membrane proteins in heterogeneous liposome populations.

Studies 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 considering 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, 25-30 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. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

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

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.

The FtsLB subcomplex of the bacterial divisome is a tetramer with an uninterrupted FtsL helix linking the transmembrane and periplasmic regions.

In Escherichia coli, FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here, we present a structural analysis of the FtsLB complex, performed with biophysical, computational, and in vivo methods, that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis in vitro is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain probably terminates near the critical constriction control domain, which might correspond to a structural transition. The presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions and that the integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

Cellular encoding of Cy dyes for single-molecule imaging.

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

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.

Raft-based interactions of gangliosides with a GPI-anchored receptor.

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.