Function Of Integral Membrane Proteins Research Articles

Modeling Alternative Conformational States of Pseudo-Symmetric Solute Carrier Transporters using Methods from Machine Learning.

The Solute Carrier (SLC) superfamily of integral membrane proteins function to transport a wide array of solutes across the plasma and organelle membranes. SLC proteins also function as important drug transporters and as viral receptors. Despite being classified as a single superfamily, SLC proteins do not share a single common fold classification; however, most belong to multi-pass transmembrane helical protein fold families. SLC proteins populate different conformational states during the solute transport process, including outward open, intermediate (occluded), and inward open conformational states. For some SLC fold families this structural "flipping" corresponds to swapping between conformations of their N-terminal and C-terminal symmetry-related sub-structures. Conventional AlphaFold2 or Evolutionary Scale Modeling methods typically generate models for only one of these multiple conformational states of SLC proteins. Here we describe a fast and simple approach for modeling multiple conformational states of SLC proteins using a combined ESM - AF2 process. The resulting multi-state models are validated by comparison with sequence-based evolutionary co-variance data (ECs) that encode information about contacts present in the various conformational states adopted by the protein. We also explored the impact of mutations on conformational distributions of SLC proteins modeled by AlphaFold2 using both conventional and enhanced sampling methods. This approach for modeling conformational landscapes of pseudo-symmetric SLC proteins is demonstrated for several integral membrane protein transporters, including SLC35F2 the receptor of a feline leukemia virus envelope protein required for viral entry into eukaryotic cells.

α-Synuclein Promotes Neuronal Dysfunction and Death by Disrupting the Binding of Ankyrin to β-Spectrin.

α-Synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. We show that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders, we demonstrate that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na+/K+ ATPase, is mislocalized when human α-synuclein is expressed in Drosophila Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. We examine the same pathway in human neurons and find that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na+/K+ ATPase, and membrane potential depolarization. Our findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies lead to neuronal dysfunction and death.SIGNIFICANCE STATEMENT The small synaptic vesicle associate protein α-synuclein plays a critical role in the pathogenesis of Parkinson's disease and related disorders, but the disease-relevant binding partners of α-synuclein and proximate pathways critical for neurotoxicity require further definition. We show that α-synuclein binds directly to β-spectrin, a key cytoskeletal protein required for localization of plasma membrane proteins and maintenance of neuronal viability. Binding of α-synuclein to β-spectrin alters the organization of the spectrin-ankyrin complex, which is critical for localization and function of integral membrane proteins, including Na+/K+ ATPase. These finding outline a previously undescribed mechanism of α-synuclein neurotoxicity and thus suggest potential new therapeutic approaches in Parkinson's disease and related disorders.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs).

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Identification of open conformation of a pLGIC stabilized by outer leaflet phospholipid

Phospholipids are the principal lipid component of plasma membranes and modulate the function of integral membrane proteins including pentameric ligand-gated ion channels (pLGIC). For example, anionic phospholipids are positive modulators of pLGIC function. Though this modulatory effect has been studied for several decades the structural mechanism has remained largely elusive. Using cryoEM and stopped-flow measurements of ion channel function in asymmetric proteoliposomes, we determine the anionic phospholipid 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG) positively modulates the pLGIC, ELIC, by binding to an outer leaflet site in the M4-M3-M1 grove.

Sampling protein-lipid interactions with swarms of trajectories and a transition matrix analysis

Lipid-protein interactions are critical for integral membrane protein function, where lipids can play the role of both solvent and ligand. Because resolving the lipids that solvate a membrane protein is experimentally challenging, simulations have come to play a prominent role in linking lipid solvation and membrane protein function. However, converging the lipid distribution around a membrane protein can require surprisingly long simulations, especially in cholesterol-rich mixtures where lipid diffusion and exchange at the surface of the protein can be quite slow.

Potential Role of Plasmalogens in the Modulation of Biomembrane Morphology.

Plasmalogens are a subclass of cell membrane glycerophospholipids that typically include vinyl- ether bond at the sn-1 position and polyunsaturated fatty acid at the sn-2 position. They are highly abundant in the neuronal, immune, and cardiovascular cell membranes. Despite the abundance of plasmalogens in a plethora of cells, tissues, and organs, the role of plasmalogens remains unclear. Plasmalogens are required for the proper function of integral membrane proteins, lipid rafts, cell signaling, and differentiation. More importantly, plasmalogens play a crucial role in the cell as an endogenous antioxidant that protects the cell membrane components such as phospholipids, unsaturated fatty acids, and lipoproteins from oxidative stress. The incorporation of vinyl-ether linked with alkyl chains in phospholipids alter the physicochemical properties (e.g., the hydrophilicity of the headgroup), packing density, and conformational order of the phospholipids within the biomembranes. Thus, plasmalogens play a significant role in determining the physical and chemical properties of the biomembrane such as its fluidity, thickness, and lateral pressure of the biomembrane. Insights on the important structural and functional properties of plasmalogens may help us to understand the molecular mechanism of membrane transformation, vesicle formation, and vesicular fusion, especially at the synaptic vesicles where plasmalogens are rich and essential for neuronal function. Although many aspects of plasmalogen phospholipid involvement in membrane transformation identified through in vitro experiments and membrane mimic systems, remain to be confirmed in vivo, the compiled data show many intriguing properties of vinyl-ether bonded lipids that may play a significant role in the structural and morphological changes of the biomembranes. In this review, we present the current limited knowledge of the emerging potential role of plasmalogens as a modulator of the biomembrane morphology.

Structural determinants of cholesterol recognition in helical integral membrane proteins

Cholesterol is an integral component of mammalian membranes. It has been shown to modulate membrane fluidity and dynamics and alter integral membrane protein function. However, understanding the molecular mechanisms of how cholesterol impacts protein function is complicated by limited and conflicting structural data. Because of the nature of the crystallization and cryo-EM structure determination, it is difficult to distinguish between specific and biologically relevant interactions and a nonspecific association. The only widely recognized search algorithm for cholesterol-integral-membrane-protein interaction sites is sequence based, i.e., searching for the so-called "Cholesterol Recognition/interaction Amino acid Consensus" motif. Although these motifs are present in numerous integral membrane proteins, there is inconclusive evidence to support their necessity or sufficiency for cholesterol binding. Here, we leverage the increasing number of experimental cholesterol-integral-membrane-protein structures to systematically analyze putative interaction sites based on their spatial arrangement and evolutionary conservation. This analysis creates three-dimensional representations of general cholesterol interaction sites that form clusters across multiple integral membrane protein classes. We also classify cholesterol-integral-membrane-protein interaction sites as either likely-specific or nonspecific. Information gleaned from our characterization will eventually enable a structure-based approach to predict and design cholesterol-integral-membrane-protein interaction sites.

Molecular organization in mixed SOPC and SDPC model membranes: Water permeability studies of polyunsaturated lipid bilayers

Polyunsaturated phospholipids in plasma membranes modulate order and dynamics of the lipid bilayer and influence integral membrane protein function. In this study, we investigate the effects of polyunsaturated fatty acid (PUFA) chains in phosphatidylcholine (PC) on osmotic water permeability and packing characteristics, using a droplet interface bilayer (DIB) as a model cell membrane. The lipid bilayer system is composed of mixtures of PC lipids with varying ratio of 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18: 0–18:1 PC) to 1-stearoyl-2-docosahexaenoylphosphatidylcholine (SDPC, 18:0–22:6 PC). Water permeability coefficients (Pf) were derived from the kinetics of osmotic filtration across the DIB. Confocal Raman microspectroscopy probed the structural and packing properties of the mixtures. The water transport parameters and Raman spectral analyses together indicate that Pf and aggregate ordering of a lipid bilayer are significantly influenced by the progressive addition of PUFA chains to a bilayer. The permeability rates across bilayers of the mixtures are a linear function of SDPC mol%. The mean disorder for the bilayer hydrocarbon region, as determined by Raman disorder parameter from the saturated chain, scales linearly with Pf. The linear trend for Pf versus degree of unsaturation in double-chain PC lipid bilayers is about half the value of that obtained for a single-chain lipid system, with implications for an evolutionary preference for phospholipids during the postulated biotic transition from single-chain lipid protocellular to modern cell membranes. The amassed results highlight the role of PUFA in modifying membrane order as manifest in bulk membrane physical properties, with characteristic differences depending on single-chain or double-chain systems.

Docosahexaenoic acid and the brain- what is its role?

Docosahexaenoic acid (DHA) is a 22-carbon omega 3 PUFA highly enriched in the neuronal cell membranes and rod outer segment membranes. When DHA is depleted from these cell membranes it is replaced nearly quantitatively by a 22-carbon omega 6 PUFA, docosapentaenoic acid, which has similar, but less potent, biophysical and physiological properties to DHA. It is speculated that omega 6-docosapentaenoic acid is a buffer to prevent the possible catastrophic effects of DHA depletion on brain and visual function. The primary insult from the loss of DHA from cell membrane glycerophospholipids, and replacement by omega 6-docosapentaenoic acid, is on the flexibility/compression of the membrane lipids which affects the optimal function of integral membrane proteins (receptors, voltage-gated ion channels and enzymes). This leads to effects on second messenger systems, and subsequently affects neurotransmitter concentrations due to 'weakened' signals from the initiating receptors. Remembering there are more than 80 billion neurones and many times more synaptic connections between neurons, a very small loss of "efficiency" in signal due to altered properties of membrane proteins would likely result in meaningful changes in brain and visual function. Additionally, impairment of neurotransmission could be due, in part, to sub-optimal brain energy metabolism (glucose entry into the brain), which is significantly reduced in omega 3 deficiency. Many studies report that dietary omega 3 deficiency results in changes in learning, coping with stress, behavioural changes, and responses in visual function. It is thus concluded that DHA is an essential fatty acid for optimal neuronal function.

Crystallization of a potassium ion channel and X-ray and neutron data collection.

The mechanism by which potassium ions are transported through ion channels is currently being investigated by several groups using many different techniques. Clarification of the location of water molecules during transport is central to understanding how these integral membrane proteins function. Neutrons have a unique sensitivity to both hydrogen and potassium, rendering neutron crystallography capable of distinguishing waters from K+ ions. Here, the collection of a complete neutron data set from a potassium ion channel to a resolution of 3.55 Å using the Macromolecular Neutron Diffractometer (MaNDi) is reported. A room-temperature X-ray data set was also collected from the same crystal to a resolution of 2.50 Å. Upon further refinement, these results will help to further clarify the ion/water population within the selectivity filter of potassium ion channels.

Regulation of KcsA by Anionic Phospholipids

Membrane lipids are known to modulate the structure and function of integral membrane proteins. This effect can be achieved directly, by specific binding sites on the protein surface, or indirectly, by changing the physical properties of the membrane bilayer. In the former, binding sites at the interface between two protein subunits, known as non-annular sites, have been implicated. Anionic lipids have been shown to modulate various functional properties of the KcsA K+-channel in different manners, and are required for channel function. Here, we present molecular dynamics simulations of KcsA with anionic and zwitterionic lipids bound in non-annular sites, in order to deduce the molecular mechanism of action of membrane lipids in the prototypical K+-channel.

Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations.

The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of β-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (∼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.

Gramicidin A Channel Formation Induces Local Lipid Redistribution I: Experiment and Simulation

Integral membrane protein function can be modulated by the host bilayer. Because biological membranes are diverse and nonuniform, we explore the consequences of lipid diversity using gramicidin A channels embedded in phosphatidylcholine (PC) bilayers composed of equimolar mixtures of di-oleoyl-PC and di-erucoyl-PC (dC18:1+dC22:1, respectively), di-palmitoleoyl-PC and di-nervonoyl-PC (dC16:1+dC24:1, respectively), and di-eicosenoyl-PC (pure dC20:1), all of which have the same average bilayer chain length. Single-channel lifetime experiments, molecular dynamics simulations, and a simple lipid compression model are used in tandem to gain insight into lipid redistribution around the channel, which partially alleviates the bilayer deformation energy associated with channel formation. The average single-channel lifetimes in the two-component bilayers (95 ± 10 ms for dC18:1+dC22:1 and 195 ± 20 ms for dC16:1+dC24:1) were increased relative to the single-component dC20:1 control bilayer (65 ± 10 ms), implying lipid redistribution. Using a theoretical treatment of thickness-dependent changes in channel lifetimes, the effective local enrichment of lipids around the channel was estimated to be 58 ± 4% dC18:1 and 66 ± 2% dC16:1 in the dC18:1+dC22:1 and dC16:1+dC24:1 bilayers, respectively. 3.5-μs molecular dynamics simulations show 66 ± 2% dC16:1 in the first lipid shell around the channel in the dC16:1+dC24:1 bilayer, but no significant redistribution (50 ± 4% dC18:1) in the dC18:1+dC22:1 bilayer; these simulated values are within the 95% confidence intervals of the experimental averages. The strong preference for the better matching lipid (dC16:1) near the channel in the dC16:1+dC24:1 mixture and lesser redistribution in the dC18:1+dC22:1 mixture can be explained by the energetic cost associated with compressing the lipids to match the channel’s hydrophobic length.

Anti-Salmonella Activity of Volatile Compounds of Vietnam Coriander.

Essential oil derived from the fresh leaves of Polygonum odoratum Lour was tested for their effects on a foodborne bacterium Salmonella choleraesuis subsp. choleraesuis ATCC 35640 using a broth dilution method. This essential oil showed a significant antibacterial activity against S. choleraesuis at the concentration of 200 µg/mL. Twenty-five volatile compounds were characterized from this essential oil by GC-MS, and aldehyde compounds were found abundant and accounted for more than three-fourths of the essential oil. Among the compounds characterized, dodecanal (C12 ) was the most abundant (55.5%), followed by decanal (C10 ) (11.6%). Both alkanals were effective against S. choleraesuis with the minimum growth inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 100 µg/mL. The most potent antibacterial activity against this bacterium was found with two minor compounds, dodecanol (lauryl alcohol) and 2E-dodecenal, both with each MBC of 6.25 µg/mL. Their primary antibacterial action against S. choleraesuis provably comes from their ability to function as nonionic surface-active agents (surfactants), disrupting the native function of integral membrane proteins nonspecifically. Thus, the antibacterial activity is mediated by biophysical processes. In the case of 2E-alkenals, a biochemical mechanism is also somewhat involved, depending on their alkyl chain length.

Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins.

The lipid bilayer that constitutes cell membranes imposes environmental constraints on the structure, folding and function of integral membrane proteins. The cell membrane is an enormously heterogeneous and dynamic system in its chemical composition and associated physical forces. The lipid compositions of cell membranes not only vary over the tree of life but also differ by subcellular compartments within the same organism. Even in the same subcellular compartment, the membrane composition shows strong temporal and spatial dependence on the environmental or biological cues. Hence, one may expect that the membrane protein conformations and their equilibria strongly depend on the physicochemical variables of the lipid bilayer. Contrary to this expectation, the structures of homologous membrane proteins belonging to the same family but from evolutionary distant organisms exhibit a striking similarity. Furthermore, the atomic structures of the same protein in different lipid environments are also very similar. This suggests that certain stable folds optimized for a specific function have been selected by evolution. On the other hand, there is growing evidence that, despite the overall stability of the protein folds, functions of certain membrane proteins require a particular lipid composition in the bulk bilayer or binding of specific lipid species. Here I discuss the specific and nonspecific modulation of folding, misfolding and function of membrane proteins by lipids and introduce several diseases that are caused by misfolding of membrane proteins.

DNA-based patterning of tethered membrane patches.

Solid-supported lipid bilayers are useful model systems for mimicking cellular membranes; however, the interaction of the bilayer with the surface can disrupt the function of integral membrane proteins and impede topological transformations such as membrane fusion. As a result, a variety of tethered or cushioned lipid bilayer architectures have been described, which retain the proximity to the surface, enabling surface-sensitive techniques, but physically distance the bilayer from the surface. We have recently developed a method for spatially separating a lipid bilayer from a solid support using DNA lipids. In this system, a DNA strand is covalently attached to a glass slide or SiO2 wafer, and giant unilamellar vesicles (GUVs) displaying the complement rupture to form a planar lipid bilayer tethered above the surface. However, the location of the patch is random, determined by where the DNA-GUV initially binds to its complement. To allow greater versatility and control, we sought a way to pattern tethered membrane patches. We present a method for creating spatially distinct tethered membrane patches on a glass slide using microarray printing. Surface-reactive DNA sequences are spotted onto the slide, incubated to covalently link the DNA to the surface, and DNA-GUVs patches are formed selectively on the printed DNA. By interfacing the bilayers with microfluidic flow cells, materials can be added on top of or fused into the membrane to change the composition of the bilayers. With further development, this approach would enable rapid screening of different patches in protein binding assays and would enable interfacing patches with electrical detectors.

Perspectives of solution NMR spectroscopy for structural and functional studies of integral membrane proteins

This article discusses future perspectives of solution NMR spectroscopy to study structures and functions of integral membrane proteins at atomic resolution, based on a review of recent progress in this area. Several selected examples of structure determinations, as well as functional studies of integral membrane proteins are highlighted. We expect NMR spectroscopy to make future key scientific contributions to understanding membrane protein function, in particular for large membrane protein systems with known three-dimensional structure. Such situations can benefit from the fact that functional NMR studies have substantially less limitations by molecular size than a full de novo structure determination. Therefore, the general potential for NMR spectroscopy to solve biologic key questions associated with integral membrane proteins is very promising.

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

Energy-dependent uphill transport but not energy-independent downhill transport by lactose permease (LacY) is impaired when expressed in Escherichia coli cells or reconstituted in liposomes lacking phosphatidylethanolamine (PE) and containing only anionic phospholipids. The absence of PE results in inversion of the N-terminal half and misfolding of periplasmic domain P7, which are required for uphill transport of substrates. Replacement of PE in vitro by lipids with no net charge (phosphatidylcholine (PC), monoglucosyl diacylglycerol (GlcDAG), or diglucosyl diacylglycerol (GlcGlcDAG)) supported wild type transmembrane topology of the N-terminal half of LacY. The restoration of uphill transport in vitro was dependent on LacY native topology and proper folding of P7. Support of uphill transport by net neutral lipids in vitro (PE > PC ≫ GlcDAG ≠ GlcGlcDAG provided that PE or PC contained one saturated fatty acid) paralleled the results observed previously in vivo (PE = PC > GlcDAG ≠ GlcGlcDAG). Therefore, a free amino group is not required for uphill transport as previously concluded based on the lack of in vitro uphill transport when fully unsaturated PC replaced E. coli-derived PE. A close correlation was observed in vivo and in vitro between the ability of LacY to carry out uphill transport, the native conformation of P7, and the lipid headgroup and fatty acid composition. Therefore, the headgroup and the fatty acid composition of lipids are important for defining LacY topological organization and catalytically important structural features, further illustrating the direct role of lipids, independent of other cellular factors, in defining membrane protein structure/function.

DNA-Based Patterning of Tethered Membrane Patches

Solid-supported lipid bilayers are useful model systems for mimicking cellular membranes; however, the interaction of the bilayer with the surface can disrupt the function of integral membrane proteins. As a result, many groups have introduced tethered lipid bilayers, which retain the proximity to the surface, enabling surface-sensitive techniques, but physically distance the bilayer from the surface. We have recently developed a method for spatially separating a lipid bilayer from a solid support using DNA lipids (Chung and Boxer et al., J. Struct. Biol., 2009). In this system, a DNA strand is covalently attached to a silane-modified glass slide or SiO2 wafer. The complementary DNA strand conjugated to a lipid moiety is inserted into giant unilamellar vesicles (GUVs), and the DNA-modified GUVs hybridize to the strands on the surface, inducing flattening and rupture of the GUV to a planar tethered lipid bilayer. However, the location of the patch is random, determined by where the DNA-GUV initially binds with its complement. To allow greater versatility and control, we sought a way to pattern tethered membrane patches. We present a method for creating spatially distinct tethered membrane patches on a glass slide using microarray printing. Surface-reactive DNA sequences are spotted onto the slide, incubated to covalently link the DNA to the surface, and DNA-GUVs patches are formed selectively on the printed DNA. Different DNA sequences can be printed on the same slide, creating a unique handle on each GUV patch. This handle enables the creation of patches of different lipid compositions, dyes, and/or DNA-lipid sequences in adjacent but distinct areas, and the control over the placement of the tethered lipid bilayer potentially allows interfacing with devices. This approach would also enable rapid screening of different patches in protein binding assays and as targets for membrane fusion.

A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes.