Single Protein Tracking of P450-Reductase in an Endoplasmic Reticulum Biomimetic Reveals a NADPH Dependent Interaction with the Membrane (original) (raw)
Journal of the American Chemical Society, 2017
Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured Kd values are highly dep...
Metabolic activity induces membrane phase separation in endoplasmic reticulum
Metabolic activity induces membrane phase separation in endoplasmic reticulum, 2017
Membrane phase behavior has been well characterized in model membranes in vitro under thermodynamic equilibrium state. However , the widely observed differences between biological membranes and their in vitro counterparts are placing more emphasis on nonequilibrium factors, including influx and efflux of lipid molecules. The endoplasmic reticulum (ER) is the largest cellular membrane system and also the most metabolically active organelle responsible for lipid synthesis. However, how the nonequilibrium metabolic activity modulates ER membrane phase has not been investigated. Here, we studied the phase behavior of functional ER in the context of lipid metabolism. Utilizing advanced vibrational imaging technique, that is, stimulated Raman scattering microscopy, we discovered that metabolism of palmitate, a prevalent saturated fatty acid (SFA), could drive solid-like domain separation from the presumably uniformly fluidic ER membrane, a previously unknown phenomenon. The potential of various fatty acids to induce solid phase can be predicted by the transition temperatures of their major metabolites. Interplay between saturated and unsaturated fatty acids is also observed. Hence, our study sheds light on cellular membrane biophysics by underscoring the nonequilibrium metabolic status of living cell. lipid metabolism | membrane phase | endoplasmic reticulum | Raman imaging | fatty acid L ipid bilayers enclose the boundary of life, and their diverse chemical compositions together with physical phases underlie membrane organizations and biological functions (1). Studies of in vitro model membranes have shown that, depending on chemical composition, mixture of lipids in a bilayer could either blend into a uniform phase or phase-separate into coexisting domains due to immiscibility. Three major phases have been observed in these studies: liquid disordered (L d), liquid ordered (L o), and solid ordered (S o), each representing a distinct type of bilayer organization (1, 2). However, biological membranes are fundamentally different from in vitro model systems. As a manifestation, liquid phase separation gives rise to static microdomains that can be readily observed in model membrane, yet the existence of raft domains (L o-like) in live cell has been a debate for decades due to much smaller domain size and faster dynamics (3). Many factors have been proposed to account for the unique aspects of biological membranes. One important factor is attributed to lipid–protein interaction, including the crowding effect of membrane protein as well as the intimate regulation by cytoskeleton (3, 4). Another less-considered but more fundamental factor concerns the thermodynamic state of the membrane. The living cell is an in-trinsically nonequilibrium system that constantly undergoes lipid turnover and membrane trafficking. This is especially relevant and important for the endoplasmic reticulum (ER), the largest membrane system as well as the lipid metabolic center in cell. Indeed, the ER is subject to the influx of lipid molecules synthesized from fatty acids, the efflux due to vesicle budding and secretion, and the exchange of lipids with other organelles via extensive and dynamic physical contacts (1). All of the above contribute to a fast turnover rate of ER lipids (half-life of ∼100 h or one cell division) (5, 6). With rich metabolic activity, ER membrane is easily perturbed from equilibrium by dynamic influx of nutrients. We hence hypothesize that the nonequilibrium factor, such as lipid synthesis in the ER, is capable of driving ER membrane from its poised L d phase to phase separation. Our hypothesis is motivated by recent studies and findings. Experimentally, fatty acids such as palmi-tate have been reported to promote saturated lipid synthesis and accumulation in the ER, which subsequently impair ER structural integrity (7–9). However, the ER phase behavior was not examined there. Theoretical study has also alluded to local phase separation when patches of membrane with different composition are delivered to a bilayer (10). This could be relevant to the condition of active lipid synthesis occurring in the ER. Toward testing this hypothesis, our study creates a non-equilibrium condition with the presence of lipid synthesis in ER membrane of living cells. To examine the spatial heterogeneity of membrane and interrogate its biophysical status, we employed the emerging vibrational imaging technique, that is, stimulated Raman scattering microscopy, and correlated it with lipidomics and fluorescence imaging. We found that ER membrane phase is susceptible to the modulation by metabolic activity. Remarkably, lipid synthesis derived from palmitate, a prevalent saturated fatty acid (SFA), even drives formation of solid-like domains, which Significance Membranes can adopt distinct phases. The endoplasmic reticulum (ER) is the largest membrane system inside cells and also harbors the richest metabolic activity including lipid synthesis. Unlike plasma membrane where separated " lipid raft " domains have been predicted and observed, ER membrane is thought to be uniformly fluidic. However, such understanding is based on bio-physical studies of model membrane under thermodynamic equilibrium. It remains unclear whether and how lipid synthesis activity perturbs the equilibrium and promotes phase segregation in ER membrane. Herein, we utilized coherent Raman imaging technique to track lipid synthesis and surprisingly revealed solid-like domains emerging from liquid ER membrane. Interestingly, this phenomenon can be tuned by the incoming nutrient source, demonstrating the susceptibility of ER membrane to nonequilibrium modulation.
Introducing a fluorescence-based standard to quantify protein partitioning into membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (K P). Although several methods to determine K P are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining K P , and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His 6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate K P. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive K P. Both modes were compared by determining the affinity of our standard, resulting in equivalent K P values. As observed in other systems, eGFP-His 6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His 6 standard. Hence, we determined the K P of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure K P and understand it in terms of molecules per lipid surface.
Biochemistry, 2005
The effect of nonlamellar-prone lipids, diacylglycerol (DG) and phosphatidylethanolamine (PE), on the stability of human cytochrome P450 1A2 (CYP1A2) was examined. When 100% phosphatidylcholine (PC) in standard vesicles was gradually replaced with either DG or PE, the stability of CYP1A2 increased; the incubation time-dependent destruction of spectrally detectable P450, decrease of catalytic activity, reduction of intrinsic fluorescence, and increased sensitivity to trypsin digestion were significantly alleviated. The ternary system of PC/PE/DG increased the stability of CYP1A2 more, even at lower concentrations of each nonlamellar-prone lipid, than that of the binary lipid mixture (PC/nonlamellar lipid). By incorporating the nonlamellar-prone lipids, the CYP1A2-induced increase of the surface pressure of the lipid monolayer was much higher compared to that for 100% PC. Increased surface pressure indicates a deep insertion of the protein into lipid monolayers. Nonlamellar lipids also increased the transition temperature of CYP1A2 in thermal unfolding and reduced the incubation time-dependent detachment of membrane-bound CYP1A2 from vesicles. Taken together, these results suggest that nonlamellar lipids per se and/or the phase properties of the membrane containing these lipids are important in the enhanced stability of CYP1A2 and the concomitant maintenance of catalytic activity of the protein.
Membrane protein dynamics: limited lipid control
Pmc Biophysics, 2009
Correlation of lipid disorder with membrane protein dynamics has been studied with infrared spectroscopy, by combining data characterizing lipid phase, protein structure and, via hydrogen-deuterium (H/D) exchange, protein dynamics. The key element was a new measuring scheme, by which the combined effects of time and temperature on the H/D exchange could be separated. Cyanobacterial and plant thylakoid membranes, mammalian mitochondria
Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins
International Journal of Molecular Sciences, 2021
Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins.
Determinants of specificity at the protein–lipid interface in membranes
FEBS Letters, 2010
The complexity of pro-and eukaryotic lipidomes is increasingly appreciated mainly owing to the advance of mass spectrometric methods. Biophysical approaches have revealed that the large number of lipid classes and molecular species detected have implications for the self-organizing potential of biological membranes, resulting in the formation of lateral heterogeneous phases. How membrane proteins are able to adapt specifically to their surrounding heterogeneous matrix, and whether this environment affects protein targeting and function, is therefore a matter of particular interest. Here, we review specific protein-lipid interactions, focusing on the molecular mechanisms that determine specificity at the protein-lipid interface, and on membrane proteins that require lipids as cofactors for their architecture and function.
Archives of Biochemistry and Biophysics, 2009
Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solutionexposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.
A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress
Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work provided insights into the subcellular distribution of lipids, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remained insufficiently characterized. Here we describe a method for purifying organellar membranes from yeast, MemPrep. We demonstrate the purity of our ER preparations by quantitative proteomics and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings have important implications for understanding the role of lipids in membrane protein insertion, folding, and their sorting along the secretory pathway. Application of...
Lipid-exchange in nanodiscs discloses membrane boundaries of cytochrome-P450 reductase
Chemical communications (Cambridge, England), 2018
Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2014
The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behaviorit has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid-lipid and protein-lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein-lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.
Membrane Fluidity Is a Key Modulator of Membrane Binding, Insertion, and Activity of 5-Lipoxygenase
Biophysical Journal, 2005
Mammalian 5-lipoxygenase (5-LO) catalyzes conversion of arachidonic acid to leukotrienes, potent mediators of inflammation and allergy. Upon cell stimulation, 5-LO selectively binds to nuclear membranes and becomes activated, yet the mechanism of recruitment of 5-LO to nuclear membranes and the mode of 5-LO-membrane interactions are poorly understood. Here we show that membrane fluidity is an important determinant of membrane binding strength of 5-LO, penetration into the membrane hydrophobic core, and activity of the enzyme. The membrane binding strength and activity of 5-LO increase with the degree of lipid acyl chain cis-unsaturation and reach a plateau with 1-palmitoyl-2-arachidonolyl-sn-glycero-3-phosphocholine (PAPC). A fraction of tryptophans of 5-LO penetrate into the hydrocarbon region of fluid PAPC membranes, but not into solid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine membranes. Our data lead to a novel concept of membrane binding and activation of 5-LO, suggesting that arachidonic-acid-containing lipids, which are present in nuclear membranes at higher fractions than in other cellular membranes, may facilitate preferential membrane binding and insertion of 5-LO through increased membrane fluidity and may thereby modulate the activity of the enzyme. The data presented in this article and earlier data allow construction of a model for membrane-bound 5-LO, including the angular orientation and membrane insertion of the protein.
A Unique Junctional Interface at Contact Sites Between the Endoplasmic Reticulum and Lipid Droplets
Frontiers in Cell and Developmental Biology, 2021
Lipid droplets (LDs) constitute compartments dedicated to the storage of metabolic energy in the form of neutral lipids. LDs originate from the endoplasmic reticulum (ER) with which they maintain close contact throughout their life cycle. These ER–LD junctions facilitate the exchange of both proteins and lipids between these two compartments. In recent years, proteins that are important for the proper formation of LDs and localize to ER–LD junctions have been identified. This junction is unique as it is generally believed to invoke a transition from the ER bilayer membrane to a lipid monolayer that delineates LDs. Proper formation of this junction requires the ordered assembly of proteins and lipids at specialized ER subdomains. Without such a well-ordered assembly of LD biogenesis factors, neutral lipids are synthesized throughout the ER membrane, resulting in the formation of aberrant LDs. Such ectopically formed LDs impact ER and lipid homeostasis, resulting in different types of...
The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function
Drug metabolism and disposition: the biological fate of chemicals, 2016
This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29th in Boston MA. The symposium focused on (1) the interactions of P450s with their redox partners, and (2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-cytochrome P450 reductase (CPR) and cytochrome b5. First, solution NMR was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6 - the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methodologies, showing that the proximal reg...
The catalytic function of cytochrome P450 is entwined with its membrane-bound nature
F1000Research, 2017
Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts-cytochrome P450-reductase and cytochrome b 5 -are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s) played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. FCS (Fluorescence Correlation Spectroscopy) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan GP (Generalized Polarization) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis, and offer a methodological design suited to different biological systems.
Biomimetic Models to Investigate Membrane Biophysics Affecting Lipid–Protein Interaction
Frontiers in Bioengineering and Biotechnology, 2020
Biological membranes are highly dynamic in their ability to orchestrate vital mechanisms including cellular protection, organelle compartmentalization, cellular biomechanics, nutrient transport, molecular/enzymatic recognition, and membrane fusion. Controlling lipid composition of different membranes allows cells to regulate their membrane characteristics, thus modifying their physical properties to permit specific protein interactions and drive structural function (membrane deformation facilitates vesicle budding and fusion) and signal transduction. Yet, how lipids control protein structure and function is still poorly understood and needs systematic investigation. In this review, we explore different in vitro membrane models and summarize our current understanding of the interplay between membrane biophysical properties and lipid-protein interaction, taken as example few proteins involved in muscular activity (dystrophin), digestion and Legionella pneumophila effector protein DrrA. The monolayer model with its movable barriers aims to mimic any membrane deformation while surface pressure modulation imitates lipid packing and membrane curvature changes. It is frequently used to investigate peripheral protein binding to the lipid headgroups. Examples of how lipid lateral pressure modifies protein interaction and organization within the membrane are presented using various biophysical techniques. Interestingly, the shear elasticity and surface viscosity of the monolayer will increase upon specific protein(s) binding, supporting the importance of such mechanical link for membrane stability. The lipid bilayer models such as vesicles are not only used to investigate direct protein binding based on the lipid nature, but more importantly to assess how local membrane curvature (vesicles with different size) influence the binding properties of a protein. Also, supported lipid bilayer model has been used widely to characterize diffusion law of lipids within the bilayer and/or protein/biomolecule binding and diffusion on the membrane. These membrane models continue to elucidate important advances regarding the dynamic properties harmonizing lipid-protein interaction.