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

Membrane proteins are often observed as higher-order oligomers, and in some cases in multiple stoichiometric forms, raising the question of whether dynamic oligomerization can be linked to modulation of function. To better understand this potential regulatory mechanism, there is an ongoing effort to quantify equilibrium reactions of membrane protein oligomerization directly 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. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Since labeling uncertainty is often unavoidable, we developed an approach to estimate labeling yields using the photobleaching probability distribution of an intrinsic dimeric control. By iterative fitting of an experimental dimeric photobleaching probability distribution to an expected dimer model, we estimate the fluorophore labeling yields and find agreement with direct measurements of labeling of the purified protein by UV-VIS absorbance before reconstitution. Using this labeling prediction, similar estimation methods are applied to determine the dissociation constant of reactive CLC-ec1 dimerization constructs without prior knowledge of the fluorophore labeling yield. Finally, we estimate the operational range of subunit labeling yields that allows for discrimination of monomer and dimer populations across the reactive range of mole fraction densities. Thus, our study maps out a practical method for quantifying fluorophore labeling directly from single-molecule photobleaching data, improving the ability to quantify reactive membrane protein stoichiometry in membranes.

Structural Determinants and Functional Significance of Dimerization for Osmosensing Transporter ProP in Escherichia coli.

Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.

Ion behavior in the selectivity filter of HCN1 channels.

Hyperpolarization-activated cyclic-nucleotide gated channels (HCNs) are responsible for the generation of pacemaker currents (If or Ih) in cardiac and neuronal cells. Despite the overall structural similarity to voltage-gated potassium (Kv) channels, HCNs show much lower selectivity for K+ over Na+ ions. This increased permeability to Na+ is critical to their role in membrane depolarization. HCNs can also select between Na+ and Li+ ions. Here, we investigate the unique ion selectivity properties of HCNs using molecular-dynamics simulations. Our simulations suggest that the HCN1 pore is flexible and dilated compared with Kv channels with only one stable ion binding site within the selectivity filter. We also observe that ion coordination and hydration differ within the HCN1 selectivity filter compared with those in Kv and cyclic-nucleotide gated channels. Additionally, the C358T mutation further stabilizes the symmetry of the binding site and provides a more fit space for ion coordination, particularly for Li+.

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.

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/mol 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.

Salt-Dependent Interactions between the C-Terminal Domain of Osmoregulatory Transporter ProP of Escherichia coli and the Lipid Membrane.

Osmosensing transporter ProP detects the increase in cytoplasmic cation concentration associated with osmotically induced cell dehydration and mediates osmolyte uptake into bacteria. ProP is a 12-transmembrane helix protein with an α-helical, cytoplasmic C-terminal domain (CTD) linked to transmembrane helix XII (TM XII). It has been proposed that the CTD helix associates with the anionic membrane surface to lock ProP in an inactive conformation and that the release of the CTD may activate ProP. To investigate this possible activation mechanism, we have built and simulated a structural model in which the CTD was anchored to the membrane by TM XII and the CTD helix was associated with the membrane surface. Molecular dynamics simulations showed specific intrapeptide salt bridges forming when the CTD associated with the membrane. Experiments supported the presence of the salt bridge Lys447-Asp455 and suggested a role for these residues in osmosensing. Simulations performed at different salt concentrations showed weakened CTD-lipid interactions at 0.25 M KCl and gradual stiffening of the membrane with increasing salinity. These results suggest that salt cations may affect CTD release and activate ProP by increasing the order of membrane phospholipids.