Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B₁₂. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes: A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This ‘stiffening’ effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35°C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

NH2-terminal spin labeling of human hemoglobin--spin states and temperature dependence

In order to explore fully how ligand- and temperature-induced alterations in the spin states of heme iron are related to protein readjustments, the spin label 4-isothiocyanate (I) was covalently attached at beta-93 cysteines and at NH2-terminal valines of various heme-iron ligand forms of human hemoglobin. It was found that the mobility of NH2-terminally bound spin labels depends on the magnetic moment of the heme iron. There is a an approximately linear relationship between the magnetic moment of the heme iron and the mobility of NH2-terminally bound spin labels. In accordance with our previous results, the temperature dependence of ESR spectra of spin-labeled hemoglobin suggests the temperature-induced protein conformational change in those heme-iron ligand forms that are characterized by the equilibrium of the spin states of the heme iron. The conformational change was sensed at both spin-label-binding sites: at beta-93 cysteines and at NH2-terminal valines.     

Electron spin resonance study of human alpha 1-acid glycoprotein interaction with a spin labelled steroid.

The interaction of human alpha 1-acid glycoprotein (AAG) with a corticosteroid was studied using nitroxide labeled deoxycorticosterone and electron spin resonance (ESR) spectroscopy. The ESR spectra of the spin labeled steroid in the presence of AAG could be used to characterize the ligand-protein interaction at equilibrium without the need of a separation between bound and free species. An association constant Ka of 6.10(5) M-1 at 20 degrees C and a binding capacity of one site per mole protein were found. ESR spectra recorded at equilibrium at various temperatures allowed the calculation of enthalpy and entropy variations for the steroid-protein interaction; these thermodynamic parameters exhibited a rapid change above 45 degrees C which may be related to a protein conformational modification above this temperature, as detected by circular dichroism study. The ESR spectra width could be used to define a polar character for the spin label environment in the steroid binding site of AAG and to calculate an apparent rotational correlation time of 2.8 x 10(-8) sec for the steroid-protein complex in aqueous solution at 20 degrees C. It can be concluded that spin labeling and ESR methodology is of value in the study of steroid-protein interactions of biological significance above all because it can provide direct physico-chemical information concerning the local environment of the ligand in its binding site at equilibrium.     

Occurrence of a Temperature-induced Phase Transition in Mitochondria Isolated from Apple Fruit

Mitochondria were isolated from fruit of six cultivars of apples differing in susceptibility to the physiological disorder, low temperature breakdown. The state 3 rate of succinate-dependent oxygen uptake and the motion of a spin label were measured at from 0 to 25 C. Arrhenius plots of the data showed that the apparent energy of activation of both respiration and motion of the spin label increased abruptly at low temperatures indicative of a temperature-induced phase change in the membrane lipids. The changes were detected with mitochondria from all of the cultivars, but the temperature at which the changes occurred did not correlate with the susceptibility of the cultivars to low temperature breakdown.     

Electron paramagnetic resonance reveals a large-scale conformational change in the cytoplasmic domain of phospholamban upon binding to the sarcoplasmic reticulum Ca-ATPase

We used EPR spectroscopy to probe directly the interaction between phospholamban (PLB) and its regulatory target, the sarcoplasmic reticulum Ca-ATPase (SERCA). Synthetic monomeric PLB was prepared with a single cytoplasmic cysteine at residue 11, which was then spin labeled. PLB was reconstituted into membranes in the presence or absence of SERCA, and spin label mobility and accessibility were measured. The spin label was quite rotationally mobile in the absence of SERCA, but became more restricted in the presence of SERCA. SERCA also decreased the dependence of spin label mobility on PLB concentration in the membrane, indicating that SERCA reduces PLB-PLB interactions. The spin label MTSSL, attached to Cys11 on PLB by a disulfide bond, was stable at position 11 in the absence of SERCA. In the presence of SERCA, the spin label was released and a covalent bond was formed between PLB and SERCA, indicating direct interaction of one or more SERCA cysteine residues with Cys11 on PLB. The accessibility of the PLB-bound spin label IPSL to paramagnetic agents, localized in different phases of the membrane, indicates that SERCA greatly reduces the level of interaction of the spin label with the membrane surface. We propose that the cytoplasmic domain of PLB associates with the lipid surface, and that association with SERCA induces a major conformational change in PLB in which the cytoplasmic domain is drawn away from the lipid surface by SERCA.     

氟化钠与强诱变剂联合作用前后膜蛋白巯基的变化及其与剂量和温度的关系

本文采用与膜蛋白巯基具特异性结合的马来酰亚胺氮氧自由基标记完整的健康人血红细胞膜,由此测得的ESR波谱表明,氟化钠在单独作用时与膜蛋白巯基结合,及其与重铬酸钾,丝裂霉素C或秋水仙素联合作用时增强与膜蛋白巯基的相互作用,并存在剂量效应关系.以上作用不论单独或联合的都有温度点.与对照组比较,氟化钠在单独作用时使温变点降得最多;在联合作用时重铬酸钾和丝裂霉素C降低温变点,而秋水仙素则增高.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation

Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme. The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin. EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding. Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation. Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature. Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.     

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo.     

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.     

Temperature-dependent conformation change in spin-labeled hemoglobin

Hemoglobin was spin labeled at β-93(F9)-cysteine with N-oxy-2,2,6,6-tetramelhylpiperidinylmaleimide. The inward shift of the high-field hyperfine line (ΔH_XXX) position in the ESR spectra of the Spin label was measured aS a function of temperature. One can expect that an abrupt change in the microenvironment around the tightly bound spin label will be reflected in the function ΔH_XXX(T) as a discontinuity (break point). This was shown for aquo-, azido-. nitro- and oxyhemoglobin derivatives. The presented results suggest that the microenvironment around the tightly hound spin label in those methemoglobin derivatives that exhibit the mixed-spin state of the heme iron is prone to an abrupt change above a certain ligand-specific temperature. The change in microenvironment of the spin label is probably due to a temperature-dependent change in the tertiary structure of the protein.     

ESR study of free and immobilized elastase.

Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.     

Carbonylcyanide p-trifluoro-methoxyphenylhydrazone-induced change of mitochondrial membrane structure revealed by lipid and protein spin labeling.

Structural information on the phenomena accompanying uncoupling of oxidative phosphorylation in mitochondria was obtained using lipid and protein spin labels. The event of partitioning, observed with a small lipid spin label, the 4,4-dimethyl-2,2-dipentyl-oxazolidine-3-oxide (6-N-11) has been studied. The ratio of polar/hydrophobic part of the third line of the spectra was decreased in the presence of the uncoupler carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP), probably indicating a higher proportion of hydrophobic environment of the label. Protein spin labels have been employed to study mobilities and rate of reduction of the labels. A long-chain maleimide spin label, the 3-2-(2-maleimidoethoxy)ethylcarbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl, in the presence of carbonylcyanide-p-trifluoro-methoxyphenylhydrazone revealed decreases of mobility and of the rate of reduction. Large amplification of these effects was obtained with a short-chain maleimide spin label, the 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. With this spin label, the effect of the uncoupler could be traced down to a concentration of 0.05 μm. It is concluded that both membrane lipid and protein are changed simultaneously in the uncoupling event.     

Conformational change in bacterio-opsin on binding to retinal.

The detailed mechanism of retinal binding to bacterio-opsin is important to understanding retinal pigment formation as well as to the process of membrane protein folding. We have measured the temperature dependence of bacteriorhodopsin formation from bacterio-opsin and all-trans retinal. An Arrhenius plot of the apparent second-order rate constants gives an activation energy of 11.6 +/- 0.7 kcal/mol and an activation entropy of -4 +/- 2 cal/mol deg. Comparison of the activation entropy to model compound reactions suggests that chromophore formation in bacteriorhodopsin involves a substantial protein conformational change. Cleavage of the polypeptide chain between residues 71 and 72 has little effect on the activation energy or entropy, indicating that the connecting loop between helices B and C is not involved in this conformational change.     

Modeling a spin-labeled fusion peptide in a membrane: implications for the interpretation of EPR experiments

Site-directed spin-labeling and electron paramagnetic resonance are powerful tools for studying structure and conformational dynamics of proteins, especially in membranes. The position of the spin label is used as an indicator of the position of the site to which it is attached. The interpretation of these experiments is based on the assumptions that the spin label does not affect the peptide configuration and that it has a fixed orientation and distance with respect to the protein backbone. Here, the validity of these assumptions is examined through implicit membrane molecular dynamics simulations of the influenza hemagglutinin fusion peptide that has been labeled with methanethiosulfonate spin label. We find that the methanethiosulfonate spin label can occasionally induce peptide orientations that differ from those adopted by the wild-type peptide. Furthermore, the spin-label resides, on average, several Angstroms deeper in the membrane than the corresponding backbone C(alpha)-atom even at sites pointing toward the solvent. The nitroxide spin label exhibits flexibility and adopts various configurations depending on the surrounding residues.     

Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein

Nitroxide spin labels were incorporated into selected sites within the β-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Cα carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.     

ESR Studies on Lipid-protein Interaction in Gluten

The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4,4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.     

Mechanism of activation of protein kinase I from rabbit skeletal muscle. Mapping of the cAMP site by spin-labeled cyclic nucleotides.

Binding of adenosine 3':5'-monophosphate (cAMP) to protein kinase (type I) from rabbit skeletal muscle has been investigated using spin-labeled cAMP derivatives. Different compounds were synthesized with the spin label attached by spacer chains of different length at different positions on the adenine base. Immobilization of the spin label, determined by comparing the electron-spin resonance spectra recorded in the presence of the kinase with those of the free ligand in solutions of different viscosities, gave information about the geometry of the cAMP site. Strong immobilization of the N-6 substituents up to a spacer length of seven atoms indicates a rather deep cleft of the cAMP site. The depth of this cleft differs, however, when the spin label is attached to the different positions at the adenine (N-6, C-2 and C-8). Whereas the N-6 derivatives indicate a rather deep site, the C-2 derivatives reveal a significantly smaller depth and C-8 substituents (syn conformation) obviously occupy a very shallow surface with almost no immobilation. In addition the binding affinities of the spin-labeled cAMP derivatives have been determined, together with those of a series of (diamagnetic) C-2 derivatives bearing hydrophobic alkyl chains of different length. The latter results helped to clarify the differences between the regions near to C-2 and N-6, respectively, of the cAMP site. N-6 spin-labeled derivatives have also been investigated in the presence of ATP and protein kinase. These results are interpreted as indicative of a conformational change at the cAMP site upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.