Biology subjects

Robertson, J. L.

Publications and source records attributed to Robertson, J. L..

10 recordsLinked to original sources

bioRxiv2025

A LARGE-SCALE EVOLUTIONARY AND STRUCTURAL ANALYSIS OF CLC CHANNELS AND TRANSPORTERS

The CLC family of membrane transport proteins consists of chloride channels and anion/proton antiporters that share a similar structural scaffold. How the same fold accommodates two fundamentally distinct mechanisms is poorly understood, and while the current set of experimental structures provide some information, the changes appear limited. In this study, we show that it is possible to scale up the structural information available using AlphaFold2 predictions and identify additional structural differences associated with each mechanistic class. A phylogenetic analysis is carried out across all known CLC genes to expand the classification to include 569 channel and 1,051 transporter homologs that have been modeled. Using distance matrices, we validate AlphaFold2s ability to detect subtle structural differences among experimentally determined CLCs and use a random forest classifier to predict CLC channel vs. transporter sub-types to learn the structural changes that are the most important in the decision. The structural changes identified overlap with and contextualize the changes observed in experimental structures, expanding structural information across sequence space. The highest ranked change includes a contraction of distances between dimerization interface helices H, I, P & Q relative to the subunit core in the channel sub-types. This study lays out an approach for quantitative, large-scale structural analyses beyond experimental data and paves the way towards structural studies expanding on different conformational states and other protein families.

bioinformatics↗

bioRxiv2024

The C-terminal activating domain promotes Panx1 channel opening

Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of ATP from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C-terminus promotes the opening of the Panx1 channel by unplugging the pore. However, this simple pore- plugging mechanism alone cannot account for the observation that a Panx1 construct ending before the caspase cleavage site remains closed. Here, we show that a helical region located immediately before the caspase cleavage site, referred to as the "C-terminal activating domain (CAD)," plays a pivotal role in facilitating Panx1 activation. Electrophysiology and mutagenesis studies uncovered that two conserved leucine residues within the CAD plays a pivotal role. Cryo- EM analysis of the construct ending before reaching the CAD demonstrated that the N-terminus extends into an intracellular pocket. In contrast, the construct including the CAD revealed that this domain occupies the intracellular pocket, causing the N-terminus to flip upward within the pore. Analysis of electrostatic free energy landscape in the closed conformation indicated that the intracellular side of the ion permeation pore may be occupied by anions like ATP, creating an electrostatic barrier for anions attempting to permeate the pore. When the N-terminus flips up, it diminishes the positively charged surface, thereby reducing the drive to accumulate anions inside the pore. This dynamic change in the electrostatic landscape likely contributes to the selection of permeant ions. Collectively, these experiments put forth a novel mechanism in which C-terminal cleavage liberates the CAD, causing the repositioning of the N-terminus to promote Panx1 channel opening.

biophysics↗

bioRxiv2023

Mitigation of membrane morphology defects explain stability and orientational specificity of CLC dimers

Most membrane proteins are oligomers, but the physical forces explaining the stable association of these complexes inside the lipid bilayer are not well understood. The homodimeric antiporter CLC-ec1 highlights the puzzling nature of this reaction. This complex is thermodynamically stable even though it associates via a large hydrophobic protein-protein interface that appears well adapted to interact with the membrane interior. In a previous study, however, we discovered that when CLC-ec1 is dissociated, this interface introduces a morphological defect in the surrounding membrane, leading us to hypothesize association is driven by the elimination of this defect upon dimerization. This study tests this hypothetical mechanism directly and shows it is supported by molecular and physical models. First, using coarse-grained umbrella-sampling molecular simulations, we calculated the membrane contribution to the potential-of-mean-force for dimerization in a POPC bilayer. This shows the stable association of CLC subunits prior to formation of direct protein-protein contacts, but only via the native interface that presents the membrane defect, and not others. Single-molecule photobleaching experiments show that addition of short-chain DLPC lipids, known to alleviate the membrane defect, also shifts the association equilibrium from dimers to monomers. We explain this destabilizing effect through additional umbrella-sampling and alchemical free-energy simulations, which show DLPC enrichment of the defect diminishes the membrane contribution to the association free energy, as it improves the lipid-solvation energetics of the monomer but not the dimer. In summary, this study establishes a physical model that explains the stability and orientational specificity of CLC dimers in terms of membrane-mediated forces, rather than protein-protein interactions. We posit that cells might ubiquitously leverage morphological defects in the bilayer to drive organization of membrane proteins into functional complexes, and that cellular regulation of lipid composition can modulate this organizing effect.

biophysics↗

bioRxiv2023

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,{Delta} G{degrees}. To ensure that the reaction reached equilibrium under changing conditions, we utilized a Forster 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.

biophysics↗

bioRxiv2023

Dimerization mechanism of an inverted-topology ion channel in membranes

Many ion channels are multi-subunit complexes with a polar permeation pathway at the oligomeric interface, but their mechanisms of assembly into functional, thermodynamically stable units within the membrane are largely unknown. Here we characterize the assembly of the inverted-topology, homodimeric fluoride channel Fluc, leveraging a known mutation, N43S, that weakens Na+ binding to the dimer interface, thereby unlocking the complex. While single-channel recordings show Na+ is required for activation, single-molecule photobleaching and bulk Forster Resonance Energy Transfer experiments in lipid bilayers demonstrate that N43S Fluc monomers and dimers exist in dynamic equilibrium, even without Na+. Molecular dynamics simulations indicate this equilibrium is dominated by a differential in the lipid-solvation energetics of monomer and dimer, which stems from hydrophobic exposure of the polar ion pathway in the monomer. These results suggest a model wherein membrane-associated forces induce channel assembly while subsequent factors, in this case Na+ binding, result in channel activation. TeaserMembrane morphology energetics foster inverted-topology Fluc channels to form dimers, which then become active upon Na+ binding.

biophysics↗

bioRxiv2022

A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes

Membrane proteins are often structured as higher-order oligomers. Yet, the role of these specific assemblies is not always apparent, raising the question of whether differential oligomerization states can be linked to modulation of function. To better understand this hypothetical regulatory mechanism, there is an ongoing effort to quantify equilibrium reactions of membrane proteins in membranes. Single-molecule photobleaching analysis is particularly useful for this as it provides a binary readout of fluorophores attached to protein subunits at dilute conditions. The subunit capture method adds consideration of the Poisson probability of protein partitioning into liposomes from large equilibrium membranes. If the liposome size distribution is known, then the capture statistics can be modeled with accuracy to quantify oligomerization as a function of membrane density to obtain binding isotherms, as was demonstrated for the dimeric chloride/proton antiporter CLC-ec1. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Since labeling uncertainty is often unavoidable, we tested an alternate approach to estimate labeling yields using the photobleaching probability of an intrinsic dimeric control, the disulfide cross-linked R230C/L249C CLC-ec1. By iterative fitting of the experimental dimeric photobleaching probability distribution to a dimer model while varying labeling parameters, we predict the labeling yields measured by direct absorbance measurements of the purified protein before reconstitution. Finally, the average predicted labeling yield over multiple samples is used to estimate the dissociation constant of CLC-ec1 dimerization reactions, eliminating the need to quantify fluorophore labeling a priori. This approach can be generalized to study dimerization reactions where an irreversible dimeric control can be prepared. Thus, our study maps out a new method for quantifying fluorophore occupancy in samples that cannot be purified directly and improves quantification of membrane protein stoichiometry in membranes.

biophysics↗

bioRxiv2020

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

biophysics↗

bioRxiv2020

Dimerization of a membrane transporter is driven by differential energetics of lipid solvation 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 caused by 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 towards 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 proteins 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. IMPACT STATEMENTDifferences in the lipid solvation energetics of associated and dissociated states is a primary driving force for membrane protein oligomerization, presenting a molecular mechanism for lipid regulation in biology.

biophysics↗

bioRxiv2019

Occupancy distributions of membrane proteins in heterogeneous liposome populations

Measurements 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.

biophysics↗

bioRxiv2017

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

We 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{Delta} G{degrees} values, providing an alternate model-free approach to measuring CLC-ec1 dimerization in membranes.

biochemistry↗