Spin Labeling EPR Spectroscopy Research Articles

Mapping α‐helical induced folding within the intrinsically disordered C‐terminal domain of the measles virus nucleoprotein by site‐directed spin‐labeling EPR spectroscopy

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.

Few residues within extensive binding interface determine receptor preference of arrestin proteins

The “footprint” of light‐activated phosphorhodopsin (P‐Rh*) on rod arrestin covers much the concave surfaces of the two domains. We used site‐directed spin labeling EPR spectroscopy to map the receptor‐binding surface of non‐visual arrestin2. The mobility of 15 residues (out of 21 tested) was reduced upon receptor binding, indicating that these positions are at or near the interaction interface. The receptor‐binding site in arrestin2 is as extensive as the rhodopsin‐binding site of rod arrestin. Taking advantage of a 4‐5‐fold difference between visual arrestin and arrestin2 binding to P‐Rh*, we used a gain‐of‐function approach to mimic rod‐like arrestin on the background of arrestin2. The combination of 5 and 8 mutations of exposed residues in the N‐ and C‐domains, respectively, corresponding to those present in rod arrestin reverses receptor preference of arrestin2. The resulting mutant binds P‐Rh* as well as wild type rod arrestin. Subsequent analysis by “loss‐of‐acquired‐function” approach, in which we mutated individual residues back to those present in arrestin2, identified one position in the N‐domain and three in the C‐domain that are primarily responsible for receptor discrimination. Thus, out of >40 amino acids in the ∼20x60A interaction interface, four exposed residues largely determine receptor specificity of arrestin proteins. NIH grants EY11500, GM 77561 (VVG), AI58024, GM70642 (CSK).

Topography of the Prostaglandin Endoperoxide H2 Synthase-2 in Membranes

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.

Electron Paramagnetic Resonance Studies of an Integral Membrane Peptide Inserted into Aligned Phospholipid Bilayer Nanotube Arrays

This communication reports for the first time the determination of the helical tilt of an integral membrane peptide inserted into aligned phospholipids bilayer nanotube arrays using spin label EPR spectroscopy. Also, we demonstrate herein how the helical tilt of the peptide can be easily calculated using the hyperfine splitting values gleaned from a perpendicularly aligned bilayer in phospholipid bilayer nanotube arrays. EPR spectral simulations were used to verify the method.

Effect of cholesterol on the interaction of seminal plasma protein, PDC-109 with phosphatidylcholine membranes

Binding of PDC-109, the major protein of the bovine seminal plasma, to sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, a necessary event before capacitation can occur. The selectivity of PDC-109 for different spin-labelled phospholipids and sterol probes in dimyristoylphosphatidylcholine (DMPC) host matrix has been characterized earlier by EPR spectroscopy [Ramakrishnan, M., Anbazhagan, V., Pratap, T.V., Marsh, D. and Swamy, M.J. (2001) Biophys. J. 81, 2215–2225]. In this report the effect of cholesterol on the interaction of PDC-109 with DMPC membranes has been investigated by spin-label EPR spectroscopy. The results indicate that the presence of cholesterol leads to an increased association of different phospholipid as well as sterol probes, thus modulating the interaction of PDC-109 with phospholipid membranes.

Quantitative Characterization of Membrane Binding of Peripheral Proteins by Spin-Label EPR Spectroscopy

Key enzymes involved in transmembrane signaling and lipid metabolism (e.g., protein kinase C and phospholipases A2, C, and D) are activated by binding to cellular membranes. Elucidation of the molecular mechanisms of these peripheral membrane proteins requires detailed characterization of their interactions with membrane lipids. Previously, EPR studies on protein−membrane interactions have been analyzed using a formalism for integral membrane proteins, permitting determination of the lipid-to-protein stoichiometry (N) and the relative affinity of the labeled versus unlabeled lipids for the protein (Kr). Here, a formalism is developed that permits a comprehensive description of the membrane binding of peripheral proteins. The interaction of an interfacially activated enzyme, secretory phospholipase A2 (PLA2), with membranes containing spin-labeled lipids is studied by EPR spectroscopy. Under noncatalytic conditions, binding of PLA2 to fluid membranes (order parameter Szz ≈ 0.24) causes the formation of a s...

Dipalmitoylphosphatidylcholine modulates inflammatory functions of monocytic cells independently of mitogen activated protein kinases.

Phosphatidylcholine (PC) is the major phospholipid of pulmonary surfactant and it is hypothesized that PC and its subspecies modulate the functions of alveolar macrophages. The most abundant of these subspecies is dipalmitoylphosphatidylcholine (DPPC). This study was undertaken to determine the effect of PC on monocyte function using a human monocytic cell line, MonoMac-6 (MM6). This study showed that preincubation of MM6 cells with DPPC at 125 microg/ml for 2 h inhibited the oxidative response to either zymosan or phorbol-12-myristate-13-acetate (PMA) by 30% (P < 0.001). This inhibition with DPPC was independent of LPS priming. When DPPC was replaced with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) there was no inhibition and in contrast a significant increase in oxidant production was observed. We also demonstrated that total PC (tPC; a heterogeneous species of PC from egg) and DPPC but not PAPC significantly inhibited the release of TNF-alpha from MM6 cells (P < 0.05). DPPC did not inhibit phosphorylation of the mitogen activated protein kinases (MAPKs) p44/p42 or p38 in stimulated cells. Measurements of membrane fluidity with spin label EPR spectroscopy indicate that DPPC incorporation significantly alters the membrane fluidity of MM6 cells. These results suggest that DPPC, the major component of pulmonary surfactant, may play a role in modulating leucocyte inflammatory responses in the lung. This may in part be related to membrane effects but does not include alterations in p44/p42 or p38 MAPK signalling.

Light-induced Rotation of a Transmembrane α-Helix in Bacteriorhodopsin

Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR). Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane α-helices B, F, and G in order to monitor distance changes upon light activation. The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle. The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin. The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins. Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base.

Phospholamban remains associated with the Ca2+- and Mg2+-dependent ATPase following phosphorylation by cAMP-dependent protein kinase.

We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca(2+)- and Mg(2+)-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl ('ITC-TEMPO'), we have co-reconstituted PLB with affinity-purified Ca-ATPase isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca(2+)-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a-PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca(2+)-transport function.

Phospholamban remains associated with the Ca2+- and Mg2+-dependent ATPase following phosphorylation by cAMP-dependent protein kinase

We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca2+- and Mg2+-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl (‘ITC-TEMPO’), we have co-reconstituted PLB with affinity-purified Ca-ATPase isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca2+-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a–PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca2+-transport function.

Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes.

We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.

Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives

Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.

Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy

We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.