G Protein-Coupled Receptor Dimerization-What Next?

Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.

Beyond the G protein α subunit: investigating the functional impact of other components of the Gαi3 heterotrimers.

BACKGROUND: Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gß and Gγ subunits and the effects of different Gßγ dimer compositions on GPCR signaling are poorly understood. In this study, we aimed to investigate the subcellular localization and functional response of Gαi3-based heterotrimers with different combinations of Gß and Gγ subunits. METHODS: Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi3 in combination with Gß1 or Gß2 heterotrimers, along with representative Gγ subunits. Furthermore, fluorescence lifetime imaging microscopy (FLIM-FRET) was used to investigate the nanoscale distribution of Gαi3-based heterotrimers in the plasma membrane, specifically with the dopamine D2 receptor (D2R). In addition, the functional response of the system was assessed by monitoring intracellular cAMP levels and conducting bioinformatics analysis to further characterize the heterotrimer complexes. RESULTS: Our results show that Gαi3 heterotrimers mainly localize to the plasma membrane, although the degree of colocalization is influenced by the accompanying Gß and Gγ subunits. Heterotrimers containing Gß2 showed slightly lower membrane localization compared to those containing Gß1, but certain combinations, such as Gαi3ß2γ8 and Gαi3ß2γ10, deviated from this trend. Examination of the spatial arrangement of Gαi3 in relation to D2R and of changes in intracellular cAMP level showed that the strongest functional response is observed for those trimers for which the distance between the receptor and the Gα subunit is smallest, i.e. complexes containing Gß1 and Gγ8 or Gγ10 subunit. Deprivation of Gαi3 lipid modifications resulted in a significant decrease in the amount of protein present in the cell membrane, but did not always affect intracellular cAMP levels. CONCLUSION: Our studies show that the composition of G protein heterotrimers has a significant impact on the strength and specificity of GPCR-mediated signaling. Different heterotrimers may exhibit different conformations, which further affects the interactions of heterotrimers and GPCRs, as well as their interactions with membrane lipids. This study contributes to the understanding of the complex signaling mechanisms underlying GPCR-G-protein interactions and highlights the importance of the diversity of Gß and Gγ subunits in G-protein signaling pathways. Video Abstract.

The Gαi protein subclass selectivity to the dopamine D2 receptor is also decided by their location at the cell membrane.

BACKGROUND: G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins plays an important role in the cellular regulation of responses to external stimuli. Despite intensive structural research, the mechanism underlying the receptor-G protein coupling of closely related subtypes of Gαi remains unclear. In addition to the structural changes of interacting proteins, the interactions between lipids and proteins seem to be crucial in GPCR-dependent cell signaling due to their functional organization in specific membrane domains. In previous works, we found that Gαs and Gαi3 subunits prefer distinct types of membrane-anchor lipid domains that also modulate the G protein trimer localization. In the present study, we investigated the functional selectivity of dopamine D2 long receptor isoform (D2R) toward the Gαi1, Gαi2, and Gαi3 subunits, and analyzed whether the organization of Gαi heterotrimers at the plasma membrane affects the signal transduction. METHODS: We characterized the lateral diffusion and the receptor-G protein spatial distribution in living cells using two assays: fluorescence recovery after photobleaching microscopy and fluorescence resonance energy transfer detected by fluorescence-lifetime imaging microscopy. Depending on distribution of data differences between Gα subunits were investigated using parametric approach-unpaired T-test or nonparametric-Mann-Whitney U test. RESULTS: Despite the similarities between the examined subunits, the experiments conducted in the study revealed a significantly faster lateral diffusion of the Gαi2 subunit and the singular distribution of the Gαi1 subunit in the plasma membrane. The cell membrane partitioning of distinct Gαi heterotrimers with dopamine receptor correlated very well with the efficiency of D2R-mediated inhibition the formation of cAMP. CONCLUSIONS: This study showed that even closely related subunits of Gαi differ in their membrane-trafficking properties that impact on their signaling. The interactions between lipids and proteins seem to be crucial in GPCR-dependent cell signaling due to their functional organization in specific membrane domains, and should therefore be taken into account as one of the selectivity determinants of G protein coupling. Video abstract.

Bradykinin B2 and dopamine D2 receptors form a functional dimer.

In recent years a wide range of studies have shown that G protein-coupled receptors modulate a variety of cell functions through the formation of dimers. For instance, there is growing evidence for the dimerization of bradykinin or dopamine receptors, both as homodimers and heterodimers. A discovery of direct interactions of angiotensin II receptors with bradykinin 2 receptor (B2R) or dopamine D2 (D2R) receptor has led to a hypothesis on a potential dimerization between two latter receptors. In this study, we have demonstrated a constitutive colocalization of receptors on the membranes of HEK293 cells transiently transfected with plasmid vectors encoding B2R and D2R, fused with fluorescent proteins. The receptor colocalization was significantly enhanced by specific agonists of B2R or D2R after 5min following the addition, whereas simultaneous stimulation with these agonists did not influence the B2R/D2R colocalization level. In addition, B2R-D2R heterodimerization was confirmed with FLIM-FRET technique. The most characteristic signaling pathways for B2R and D2R, dependent on intracellular Ca2+ and cAMP concentration, respectively, were analyzed in cells presenting similar endogenous expression of B2R and D2R. Significant changes in receptors' signaling were observed after simultaneous stimulation with agonists, suggesting transformations in proteins' conformation after dimerization. The evidence of B2R-D2R dimerization may open new perspectives in the modulation of diverse cellular functions which depend on their activation.

The role of cholesterol and sphingolipids in the dopamine D1 receptor and G protein distribution in the plasma membrane.

G proteins are peripheral membrane proteins which interact with the inner side of the plasma membrane and form part of the signalling cascade activated by G protein-coupled receptors (GPCRs). Since many signalling proteins do not appear to be homogeneously distributed on the cell surface, they associate in particular membrane regions containing specific lipids. Therefore, protein-lipid interactions play a pivotal role in cell signalling. Our previous results showed that although Gαs and Gαi3 prefer different types of membrane domains they are both co-localized with the D1 receptor. In the present report we characterize the role of cholesterol and sphingolipids in the membrane localization of Gαs, Gαi3 and their heterotrimers, as well as the D1 receptor. We measured the lateral diffusion and membrane localization of investigated proteins using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by lifetime imaging microscopy (FLIM). The treatment with either methyl-ß-cyclodextrin or Fumonisin B1 led to the disruption of cholesterol-sphingolipids containing domains and changed the diffusion of Gαi3 and the D1 receptor but not of Gαs. Our results imply a sequestration of Gαs into cholesterol-independent solid-like membrane domains. Gαi3 prefers cholesterol-dependent lipid rafts so it does not bind to those domains and its diffusion is reduced. In turn, the D1 receptor exists in several different membrane localizations, depending on the receptor's conformation. We conclude that the inactive G protein heterotrimers are localized in the low-density membrane phase, from where they displace upon dissociation into the membrane-anchor- and subclass-specific lipid domain.

Intra- and intersubunit changes accompanying thermal activation of the HtrA2(Omi) protease homotrimer.

HtrA2(Omi) protease is involved in the maintenance of mitochondrial homeostasis and stimulation of apoptosis as well as in development of cancer and neurodegenerative disorders. The protein is a homotrimer whose subunits comprise serine protease domain (PD) and PDZ regulatory domain. In the basal, inactive state, a tight interdomain interface limits access both to the PDZ peptide (carboxylate) binding site and to the PD catalytic center. The molecular mechanism of activation is not well understood. To further the knowledge of HtrA2 thermal activation we monitored the dynamics of the PDZ-PD interactions during temperature increase using tryptophan-induced quenching (TrIQ) method. The TrIQ results suggested that during activation the PDZ domain changed its position versus PD inside a subunit, including a prominent change affecting the L3 regulatory loop of PD, and also changed its interactions with the PD of the adjacent subunit (PD*), specifically with its L1* regulatory loop containing the active site serine. The α5 helix of PDZ was involved in both, the intra- and intersubunit changes of interactions and thus seems to play an important role in HtrA2 activation. The amino acid substitutions designed to decrease the PDZ interactions with the PD or PD* promoted protease activity at a wide range of temperatures, which supports the conclusions based on the TrIQ analysis. The model presented in this work describes PDZ movement in relation to PD and PD*, resulting in an increased access to the peptide binding and active sites, and conformational changes of the L3 and L1* loops.

Comparative EPR studies on lipid bilayer properties in nanodiscs and liposomes.

Studies of the membrane proteins suggest their close interaction with the lipid surroundings. Membrane proteins and their activities are affected by the composition and structure of the lipid bilayer. therefore adequate surroundings for studied protein are crucial for the model membrane to ensure its biological relevance. In recent years nanodiscs which are small fragments of lipid bilayer stabilised by derivatives of apolipoprotein, called membrane scaffold protein ( MSP), have been established as alternative tool in structural and functional studies of membrane proteins. In this study, the influence MSP of different length on structure and dynamics of DMPC and POPC bilayer was investigated and compared to bilayer present in liposomes. EPR spectroscopy technique using different PC-based spin probes was employed to show cholesterol-like organising effect of MSPs on lipid bilayer, thus giving a better insight into the nanodiscs model membrane structure, and its possible implications in the research of membrane protein applications.

New insights into the model of dopamine D1 receptor and G-proteins interactions.

The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane. Using fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) or fluorescence recovery after photobleaching (FRAP) microscopy, we did not detect constitutive preassociated complex between D1 receptor and G-protein in the absence of receptor activation. Our work suggests that D1 receptor alters the distribution of Gαs and Gαi3 subunits inside the membrane. We also find that non-activated D1 receptor and Gαs or Gαi3 are present in the cell membrane within the same membrane microdomains in the proximity of about 9-10 nm.

The LA loop as an important regulatory element of the HtrA (DegP) protease from Escherichia coli: structural and functional studies.

Bacterial HtrAs are serine proteases engaged in extracytoplasmic protein quality control and are required for the virulence of several pathogenic species. The proteolytic activity of HtrA (DegP) from Escherichia coli, a model prokaryotic HtrA, is stimulated by stressful conditions; the regulation of this process is mediated by the LA, LD, L1, L2, and L3 loops. The precise mechanism of action of the LA loop is not known due to a lack of data concerning its three-dimensional structure as well as its mode of interaction with other regulatory elements. To address these issues we generated a theoretical model of the three-dimensional structure of the LA loop as per the resting state of HtrA and subsequently verified its correctness experimentally. We identified intra- and intersubunit contacts that formed with the LA loops; these played an important role in maintaining HtrA in its inactive conformation. The most significant proved to be the hydrophobic interactions connecting the LA loops of the hexamer and polar contacts between the LA' (the LA loop on an opposite subunit) and L1 loops on opposite subunits. Disturbance of these interactions caused the stimulation of HtrA proteolytic activity. We also demonstrated that LA loops contribute to the preservation of the integrity of the HtrA oligomer and to the stability of the monomer. The model presented in this work explains the regulatory role of the LA loop well; it should also be applicable to numerous Enterobacteriaceae pathogenic species as the amino acid sequences of the members of this bacterial family are highly conserved.

The polybasic region in Gαi proteins: Relevant or not? Insights from Gαi3 research.

Heterotrimeric G proteins are responsible for signal transduction from G-protein-coupled receptors (GPCRs) to intracellular effectors. This process is only possible when G proteins are located on the inner side of the cell membrane due to the specific localization of GPCR receptors. The Gα subunit is directed to the cell membrane through several signals, including modification by fatty acid moieties, interaction with the Gßγ complex, and, as observed in some Gα proteins, the presence of basic amino acid residues in the N-terminal region. In this work, we focused on investigating the influence of the polybasic region on the localization and function of a representative member of the Gαi family, Gαi3. Through the use of confocal microscopy and fluorescence lifetime microscopy, we showed that, in the case of this protein, neutralizing the positive charge does not significantly affect its abundance in the cell membrane. However, it does affect its spatial arrangement concerning the dopamine D2 receptor and influences inhibitory effect of Gαi3 on intracellular cAMP production triggered by D2 receptor stimulation. Moreover, in this work, we have shown, for the first time, that nonlipidated Gαi3 binds to negatively charged lipids through electrostatic interactions, and membrane fluidity plays a significant role in this interaction.

The differences in binding 12-carbon aliphatic ligands by bovine ß-lactoglobulin isoform A and B studied by isothermal titration calorimetry and X-ray crystallography.

Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X-ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC < LA < SDS, and were systematically lower for variant B. The entropy change of the complexation process was always higher for isoform A, but these values were compensated by changes in enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein ß-barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance.

Hetero-dimerization of serotonin 5-HT(2A) and dopamine D(2) receptors.

In the present study, detailed information is presented on the hetero-dimerization of the serotonin 5-HT(2A) receptor and the dopamine D(2) receptor. Biophysical approaches (fluorescence spectroscopy as well as fluorescence lifetime microscopy) were used to determine the degree of fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein labeled receptor variants co-expressed in human embryonic kidney 293 cells (HEK293). Recorded data demonstrate the existence of energy transfer between the wild-type forms of 5-HT(2A)R and D(2)R, pointing toward the formation of hetero-5-HT(2A)R/D(2)R dimers and homo-5-HT(2A)R/5-HT(2A)R dimers. Moreover, the present study investigates the role of specific motifs (one containing adjacent arginine residues (217RRRRKR222) in the third intracellular loop (ic3) of D(2)R, and the other consisting of acidic glutamate residues (454EE455) in the C-tail of (5-HT(2A)R) in the formation of noncovalent complexes between these receptors. Our results suggest that these regions of 5-HT(2A)R and D(2)R may be involved in the interaction between these two proteins. On the other hand, the above-mentioned motifs do not play an important role in the homo-dimerization of these receptors. Furthermore, we estimated the influence of specific receptor ligands on the dimerization processes. Agonists (DOI and quinpirole) and antagonists (ketanserin and butaclamol) cause different effects on FRET efficiency depending on whether homo- or hetero-complexes are present. These data may have therapeutic implications, since (using the immunofluorescence double labeling protocols) the co-localization of these two receptors was demonstrated in the medial prefrontal cortex and pars reticulate of the substantia nigra of the rat brain.

Two modes of fatty acid binding to bovine ß-lactoglobulin--crystallographic and spectroscopic studies.

Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12-20 carbon atoms long. Shorter fatty acids were reported as not binding to ß-lactoglobulin. We used X-ray crystallography and fluorescence spectroscopy to show that lactoglobulin binds also 8- and 10-carbon caprylic and capric acids, however with lower affinity. The determined apparent association constant for lactoglobulin complex with caprylic acid is 10.8 ± 1.7 × 10(3) M(-1), while for capric acid is 6.0 ± 0.5 × 10(3) M(-1). In crystal structures determined with resolution 1.9 Å the caprylic acid is bound in upper part of central calyx near polar residues located at CD loop, while the capric acid is buried deeper in the calyx bottom and does not interact with polar residues at CD loop. In both structures, water molecule hydrogen-bonded to carboxyl group of fatty acid is observed. Different location of ligands in the binding site indicates that competition between polar and hydrophobic interactions is an important factor determining position of the ligand in ß-barrel.

Every Detail Matters. That Is, How the Interaction between Gα Proteins and Membrane Affects Their Function.

In highly organized multicellular organisms such as humans, the functions of an individual cell are dependent on signal transduction through G protein-coupled receptors (GPCRs) and subsequently heterotrimeric G proteins. As most of the elements belonging to the signal transduction system are bound to lipid membranes, researchers are showing increasing interest in studying the accompanying protein-lipid interactions, which have been demonstrated to not only provide the environment but also regulate proper and efficient signal transduction. The mode of interaction between the cell membrane and G proteins is well known. Despite this, the recognition mechanisms at the molecular level and how the individual G protein-membrane attachment signals are interrelated in the process of the complex control of membrane targeting of G proteins remain unelucidated. This review focuses on the mechanisms by which mammalian Gα subunits of G proteins interact with lipids and the factors responsible for the specificity of membrane association. We summarize recent data on how these signaling proteins are precisely targeted to a specific site in the membrane region by introducing well-defined modifications as well as through the presence of polybasic regions within these proteins and interactions with other components of the heterocomplex.

Can di-4-ANEPPDHQ reveal the structural differences between nanodiscs and liposomes?

The potential-sensitive di-4-ANEPPDHQ dye is presently gaining popularity in structural studies of the lipid bilayer. Within the bilayer, dye environmental sensitivity originates from the excitation induced charge redistribution and is usually attributed to solvent relaxation. Here, di-4-ANEPPDHQ is utilized to compare the structure of neutral and negatively charged lipid bilayers between two model systems: the nanodiscs and the liposomes. Using the well-established approach of measuring solvatochromic shifts of the steady-state spectra to study the bilayer structural changes has proved insufficient in this case. By applying an in-depth analysis of time-resolved fluorescence decays and emission spectra, we distinguished and characterized two and three distinct emissive di-4-ANEPPDHQ species in the liposomes and the nanodiscs, respectively. These emissive species were ascribed to the dual emission of the dye rather than to solvent relaxation. An additional, long-lived component present in the nanodiscs was associated with a unique domain of high order, postulated recently. Our results reveal that the di-4-ANEPPDHQ steady-state fluorescence should be interpreted with caution. With the experimental approach presented here, the di-4-ANEPPDHQ sensitivity was improved. We confirmed that the bilayer structure is, indeed, altered in the nanodiscs. Moreover, molecular dynamic simulations showed a distribution of the probe in the nanodiscs plane, which is sensitive to lipid composition. In POPC nanodiscs, probe frequently interacts with MSP, while in POPC-POPG nanodiscs, such interactions are rare. We did not observe, however, any impact of those interactions on the probe fluorescence.

Temperature-induced conformational changes within the regulatory loops L1-L2-LA of the HtrA heat-shock protease from Escherichia coli.

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity.

Complexity of seemingly simple lipid nanodiscs.

Lipid nanodiscs are macromolecular assemblies, where a scaffold protein is wrapped around a nanosized disc of a lipid bilayer, thus protecting the hydrocarbon chains at the disc edges from unfavorable interactions with water. These nanostructures have numerous applications in, e.g., nanotechnology and pharmaceutics, and in investigations of membrane proteins. Here, we present results based on atomistic molecular dynamics simulations combined with electron paramagnetic spectroscopy measurements on the structure and dynamics of lipids in single-component nanodiscs. Our data highlight the existence of three distinctly different lipid fractions: central lipids residing in the center of a nanodisc, boundary lipids in direct contact with a scaffold protein, and intermediate lipids between these two regions. The central lipids are highly ordered and characterized by slow diffusion. In this part of the nanodisc, the membrane is the thickest and characterized by a gel-like or liquid-ordered phase, having features common to cholesterol-rich membranes. The boundary lipids in direct contact with the scaffold protein turned out to be less ordered and characterized by faster diffusion, and they remained in the liquid-disordered phase even at temperatures that were somewhat below the main phase transition temperature (Tm). The enthalpies associated with the central-boundary and central-intermediate transitions were similar to those observed for lipids going through the main phase transition. Overall, the study reveals lipid nanodiscs to be characterized by a complex internal structure, which is expected to influence membrane proteins placed in nanodiscs.

Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting.

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various heterotrimeric complexes. In a previous study, we found that Gαs and Gαi3 prefer different types of membrane-anchor and subclass-specific lipid domains. In the present report, we examine the role of distinct Gγ subunits in the membrane localization and spatiotemporal dynamics of Gαs and Gαi3 heterotrimers. We characterized lateral diffusion and G-protein subunit interactions in living cells using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM), respectively. The interaction of Gγ subunits with specific lipids was confirmed, and thus the modulation of heterotrimeric G-protein localization. However, the Gα subunit also modulates trimer localization, and so the membrane distribution of heterotrimeric G-proteins is not dependent on Gγ only.

Cholesteryl Hemisuccinate Is Not a Good Replacement for Cholesterol in Lipid Nanodiscs.

Nanodiscs are suitable tools for studies of membrane proteins (MPs) due to their ability to mimic native biological membranes, and several MP structures are solved in nanodiscs. Among the various cell membrane components, cholesterol (CHL) is known to regulate protein function and its concentration can reach up to 50 mol %. However, studies comprising cholesterol are challenging due to its hydrophobic nature, hence, nanodiscs with only a low cholesterol concentration have been studied. To overcome the problem, cholesterol analogs with high solubility in polar solutions are often used, and one of them is cholesteryl hemisuccinate (CHS). Nevertheless, in molecular dynamics (MD) simulation, this is not an obstacle. In this study, we performed MD simulations of nanodiscs containing neutral phosphatidylcholine (POPC) lipids, negatively charged phosphatidylglycerol (POPG) lipids, CHL, or negatively charged cholesterol analog, CHS. Our simulations show that CHS increases the order of lipids in nanodiscs; the effect is, however, weaker than CHL and even smaller in nanodiscs. Furthermore, CHS gathered around scaffold proteins while cholesterol was uniformly distributed in the nanodiscs. Thus, nanodiscs with CHS are heterogeneous and not equivalent to nanodiscs with CHL. Finally, we also observed the increased concentration of POPG near the scaffold proteins, driven by electrostatic interactions. The MD results are experimentally validated using electron paramagnetic resonance spectroscopy. These results show that nanodiscs are, in fact, complex structures not easily comparable with planar lipid bilayers.

Systematic calorimetric studies of proton exchange associated with binding of beta-lactoglobulin with ligand.

In proteins, proton exchange is caused due to the changes in the proton affinity (pKa) of ionizable groups that are engaged in conformational changes induced by the binding of a ligand. In addition, knowledge regarding the type and number of such ionizable groups is very important to understand the pH-dependent changes of the thermodynamic parameters. Therefore, in this study, we performed a comprehensive analysis of proton exchange by using beta lactoglobulin (Blg), a representative of the lipocalin family of proteins. We used isothermal titration calorimetry to evaluate the proton exchange during binding with sodium dodecyl sulfate (SDS) at different pHs ranging from 2 to 9. SDS binds to Blg in all studied range of pHs and enthalpy-driven reactions are observed in acidic pH, whereas enthalpy-entropy driven reactions are observed in neutral and basic pHs. Enthalpy-entropy compensation leads to relatively small changes in the association constants with the maximal value at pH = 8.0. The simultaneous analysis of the number of exchanged protons, the binding constants, and the enthalpy was performed in the range pH 5.5-9. At least 4 residues that change their ionization pattern are needed to explain the observed pH dependence.