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The emission intensity of an indolyl maleimide derivative increases approximately 80-fold by reversible coordination to (1,4,7,11-tetraazacyclododecane)zinc(II), which makes the system a promising new signalling motif for molecular sensors.
A new synthesis procedure has been developed for a series of maleimide-based fluorophores, exhibiting a large variation of emission spectra spanning the entire visible range.
The diagnosis of hereditary spherocytosis (HS) is based on red cell morphology and other conventional tests such as osmotic fragility, autohemolysis and acidified glycerol lysis. However, milder cases are at times difficult to diagnose. Confirmation by red blood cell (RBC) membrane protein analysis is not possible in most laboratories. Recently, a flow cytometric method has been described for quantitating the fluorescence intensity of intact red cells after incubation with the dye eosin-5'-maleimide (EMA), which binds specifically to the anion transport protein (band-3) at lysine-430. This has been shown to be an effective screening test for red cell membrane disorders. We evaluated the usefulness of this approach for screening membrane protein disorders such as HS and hereditary elliptocytosis (HE) and its value in discriminating this group from other hemolytic anemias, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, beta-thalassemia trait, sickle cell anemia and autoimmune hemolytic anemia. Fluorescence intensity, expressed in mean channel fluorescence (MCF) units, was determined using a Becton Dickinson FACS Caliber flow cytometer. Membrane protein analysis was carried out by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). RBCs from patients with HS and HE gave significantly lower MCF values (P < 0.001) than the normal control group and other patient groups. The diagnosis of HS in four cases was confirmed by RBC membrane protein electrophoresis and all showed a deficiency of spectrin. The advantage of the EMA dye method are its specificity for membrane disorders, as well as being a simple, user-friendly and rapid method which is inexpensive, provided a flow cytometer is available.
Flow cytometric analysis of eosin-5-maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS). The present study used chemical modifications to determine the integral membrane proteins that react with EMA. The predominant interaction of EMA, contributing c. 80% of fluorescence, was with the epsilon-NH2 group of lysine in band 3 protein, as previously reported. The remainder of the EMA fluorescence was attributable to labelling of accessible sulfhydryl groups on intact red cells. This reaction was heat labile. Three molecules containing sulfhydryl groups were shown to be associated with the Rh blood group protein complex by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These were CD47 and the Rh-associated glycoprotein, both in the Mr 40-60 kD region, and the Rh blood group proteins in the 30-32 kD region. Immunoprecipitation, using specific monoclonal antibodies and antibody binding studies by flow cytometry, showed that the relative content of these three membrane proteins was lower in HS than normal red cells. Thus, the high predictive value of the EMA binding test for HS reflects changes in the relative amounts of the Rh-related integral membrane proteins as well as band 3 in HS red cells.
The resolution and sensitivity of electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) for biological applications are greatly improved by deuteration and substitution of (15)N for (14)N in the spin-labeled probe N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (MSL). The EPR and ST-EPR spectra of the deuterated analogue [(2)H]MSL and the (15)N-substituted and deuterated derivative [(15)N, (2)H]MSL were compared with those of the parent MSL. The [(15)N, (2)H]MSL showed the greatest gain in sensitivity and the most marked sharpening of spectral features. These improvements were due to (i) a reduction in the spectral linewidths resulting from the relatively weak hyperfine interactions of the unpaired electron with deuterium and (ii) spectral simplification due to a reduction in the number of nuclear manifolds from three to two in replacing (14)N with (15)N. In the freely tumbling state, the spectra of [(15)N, (2)H]MSL and [(2)H]MSL showed 10-fold and 5-fold increases, respectively, in signal heights compared to MSL. To study the slow tumbling frequencies characteristic of biological molecules, the MSL and its derivatives were covalently bound to the enzyme glyceraldehyde-3-phosphate dehydrogenase [GAPDHaase; D-glyceraldehyde-3-phosphate:NAD(+) oxidoreductase (phosphorylating), EC 1.2.1.12] on cysteine-149 of the catalytic site. The EPR and ST-EPR spectra of [(15)N, (2)H]MSL and [(2)H]MSL adducts showed 3- and 1.5-fold gains in sensitivity, respectively. More important, there were striking increases in resolution, particularly for [(15)N, (2)H]MSL over MSL. These improvements were observed throughout the correlation time range from 0.1 musec to 1 msec. The EPR spectrum of [(15)N, (2)H]MSL-GAPDHase at X-band showed no overlap of the two nuclear manifolds; therefore, all the elements of the A and g tensors could be measured directly from the spectrum. The increase in sensitivity and resolution of the (15)N- and deuterium-substituted spin labels permitted quantitative simulation of the EPR and ST-EPR spectra of a labeled protein. Computation time was reduced 90% by (15)N substitution. Use of (15)N-substituted and deuterated spin probes substantially improved characterization of the motional properties of a protein.
Binding of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDHase; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating EC 1.2.1.12], to the cytoplasmic segment of band-3 protein in the erythrocyte (RBC) membrane has been examined by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies. GAPDHase, which was isolated from rabbit muscle and labeled with the resolution-enhancing deuterated N-(15N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide spin label ([15N,2H]MSL), showed the same binding specificity for the transmembrane band-3 protein of human erythrocyte membranes as reported for unlabeled GAPDHase from human RBC. Experimental EPR lineshapes from soluble and membrane-bound enzymes were analyzed by direct stimulation of spectra and indicated a structural alteration of the bound GAPDHase in the vicinity of the spin label, which was attached covalently to the active-site cysteine-149 residue. A rigorous theoretical analysis of the ST-EPR spectra of soluble and membrane-bound enzyme is presented and utilized in conjunction with model system analysis to demonstrate that the motion of membrane-bound GAPDHase could be characterized by an effective isotropic rotational correlation time of 20 microseconds. This indicated that the GAPDHase--band-4 complex exhibits motional freedom relative to the membrane-spanning segment of the band-3 protein or the RBC. The double substituted spin label [15N,2H]MSL affords gains in sensitivity and resolution that permit studies of membrane-bound enzymes at physiological levels and quantitative simulations of the EPR and ST-EPR lineshapes with reasonable computation times.
A characteristic feature of the vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) of plant cells is its high sensitivity to irreversible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl reagents. Previous investigations in this laboratory have demonstrated that the primary site for substrate-protectable covalent modification of the V-PPase by 14C-labeled NEM maps to a single M(r) 14,000 V8 protease fragment (V8(14)K) (Zhen, R.-G., Kim, E. J., and Rea, P. A. (1994) J. Biol. Chem. 269, 23342-23350). Here, we describe site-directed mutagenesis of the cDNA encoding the V-PPase from Arabidopsis thaliana, its heterologous expression in Saccharomyces cerevisiae and single substitution of all 9 conserved Cys residues to either Ser or Ala. In all cases, except one, Cys mutagenesis exerts little or no effect on either the catalytic activity or susceptibility of the enzyme to inhibition by NEM. By contrast, and in complete agreement with the results of peptide mapping experiments, substitution of Cys634, the sole conserved cysteine residue encompassed by V8(14)K, with Ser or Ala generates enzyme that is insensitive to NEM but active in both PPi hydrolysis and PPi-dependent H+ translocation. The specific requirement for Cys634 for inhibition by NEM and the dispensability of all of the conserved Cys residues, including Cys634, for V-PPase function indicate that the inhibitory action of maleimides reflects steric constraints imposed by the addition of a substituted alkyl group to the side chain of Cys634 rather than direct participation of this amino acid residue in catalysis.
We have extended previous observations to show that the ATPase N-ethyl maleimide sensitive factor (NSF) an important regulator of membrane trafficking and fusion in somatic cells, is present on bovine, murine and rhesus macaque sperm. However, NSFs main effectors, alfa- and beta-SNAP, although present in the developing acrosome, could not be detected in the mature organelle. The fact that NSF localizes mainly to the acrosome suggests that this protein, together with other factors such as rabs and SNAREs, may be a common feature in the triggering/regulation of membrane merging during the mammalian acrosome reaction.
Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5-min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3-substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site.
N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.
N-(1-Oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide (MSL) was incorporated into rat liver mitochondria and the nitroxide radical incorporated was found to decay considerably. The incorporation was blocked by a high concentration of NEM, but not by pCMB. Spin labeled fatty acid derivatives, 2-(3-carboxypropyl)-2-tridecyl-4,4-dimethyl-3-oxazolidinyloxyl (FSL1) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (FSL2), were also incorporated and the nitroxide radical decayed. However, incorporation of FSL1 or FSL2 was not blocked by NEM or pCMB. The ESR spectrum of 3-carboxyl-2,2,5,5-tetramethyl-pyrroline-1-oxyl (CSL) did not change on reaction with the mitochondria. The labeled MSL exhibited an ESR spectrum composed of both strongly immobilized and weakly immobilized components. A similar reaction with FSL1 gave an ESR spectrum mainly composed of a strongly immobilized component, the weakly immobilized component was negligibly small, while FSL2 exhibited an ESR spectrum in which free-like signals of the nitroxide radical were predominant. The results suggest that MSL is labeled selectively in the mitochondrial membrane through those SH groups that are not reactive to pCMB, and the labeled nitroxide radical is reduced in situ. The mode of incorporation into the mitochondria differs between MSL and the other spin labeled reagents, and labeling of MSL at the binding site may precede reduction of the nitroxide radical. The incorporation of MSL was dependent on the concentration of MSL used. ADP-acceleration of mitochondrial oxygen uptake with succinate was inhibited by labeling the mitochondria with MSL without loss of the electron transferring activity.
Under specified UV-MALDI conditions, the 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) derivative of cysteine-containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine-containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine-containing peptides.
A method for the quality control of platelet viability, based on N-ethyl-maleimide (NEM)-induced lipid peroxidation, is reported. Duplicate platelet concentrates from individual donors were stored at 22 and 4 degrees C, respectively. At time 0 and at 24-hour intervals, pH, O2 and CO2 tensions, ADP-induced aggregation, glass adhesivity, hypotonic shock response (HSR) and NEM-induced lipid peroxidation were measured. A highly significant correlation between NEM-induced lipid peroxidation and HSR was established, which suggests that the former method can be used for the in vitro prediction of posttransfusion survival of preserved platelets.
Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS-polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochrondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.
Although eosin-5-maleimide (EM) covalently labels band 3 and has been thought to react at the external-facing anion transport site, EM reversibly inhibits Cl- exchange at 0 degrees C in a noncompetitive fashion, indicating that under these conditions it does not bind to the transport site [Knauf, P.A., N.M. Strong, J. Penikas, R.B. Wheeler, Jr., and S.J. Liu. Am. J. Physiol. 264 (Cell Physiol. 33): C1144-C1154 1993]. To see whether or not the covalent labeling by EM takes place at the same noncompetitive site as the reversible binding, we examined the dependence of reaction rate on EM concentration. The reaction rate saturates with increasing EM concentration, indicating that reversible binding precedes covalent reaction and that EM therefore acts as an affinity label. A more complex model in which reversible binding prevents a bimolecular reaction at a different site cannot, however, be ruled out. Cl- gradients across the membrane affect EM reversible binding in a manner suggesting that EM binds preferentially to the Eo form of band 3, with the transport site unloaded and facing outward. Thus EM binds to and probably reacts covalently with a site that is different from the transport site, but whose conformation is affected by the orientation of the transport site. Lysine-430, the amino acid residue which is covalently labeled by EM (4), may be near the transport site but does not seem to be directly involved in the binding of transported substrates such as chloride. EM binding to one band 3 monomer decreases the reactivity of the adjacent monomer but does not decrease the affinity constant of the reversible binding step that precedes covalent reaction. Although a small fraction (approximately 1%) of band 3 monomers fail to react with EM, EM nearly completely inhibits transport in those monomers with which it reacts.
N-(4-(7 - Diethylamino - methylcoumarin - 3 yl)phenyl)maleimide (CPM) and the corresponding iodoacetamide are described. When applied to sections at pH 6 and 9, respectively, the two fluorogens are very similar in their reactivity and selectivity toward thiols with which they form adducts having much the same bright blue fluorescence. However, difference in other properties favor CPM for further study.
The interaction between EMI (eosin-5-maleimide) and band 3 of human erythrocytes was studied under various conditions. It was found that the effects of the ionic strength on the EMI-band 3 interaction strongly depended on pH. At pH 6.0, the ionic strength had remarkable effects on the EMI-bound band 3, whereas at pH 7.4, the EMI-band 3 interaction was independent of ionic strength. From the change in the circular dichroism spectra of the EMI-bound band 3, it was revealed that the conformation or the structure of the EMI-binding sites in the cytoplasmic domain of band 3 was strongly dependent on ionic strength. The thermodynamic parameters for the covalent-binding between EMI and band 3 were calculated on the basis of the difference spectra of the EMI and ghost system. The values of activation energy and activation entropy change at pH 6.0 were extraordinarily small compared with those values at pH 7.4. These findings represent the characteristics of the EMI-binding sites in the cytoplasmic domain of band 3. The interaction of EMI with an isolated fragment of the cytoplasmic domain, 43k fragment, was also examined. The circular dichroism spectra of the EMI-bound 43k fragments was significantly different from those of the EMI-bound band 3. This may indicate that the quaternary structure of the EMI-binding site in the cytoplasmic domain of band 3 is altered by an allosteric connection with the membrane-spanning domain of band 3. Further, from the pH titration of the 43k fragment, it was suggested that lysine residues are responsible for the ionic interaction between EMI and the 43k fragment.
A single molecule fluorescence assay is presented for studying the mechanism of soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs)-mediated liposome fusion to supported lipid bilayers. The three neuronal SNAREs syntaxin-1A, synaptobrevin-II (VAMP), and SNAP-25A were expressed separately, and various dye-labeled combinations of the SNAREs were tested for their ability to dock liposomes and induce fusion. Syntaxin and synaptobrevin in opposing membranes were both necessary and sufficient to dock liposomes to supported bilayers and to induce thermally activated fusion. As little as one SNARE interaction was sufficient for liposome docking. Fusion of docked liposomes with the supported bilayer was monitored by the dequenching of soluble fluorophores entrapped within the liposomes. Fusion was stimulated by illumination with laser light, and the fusion probability was enhanced by raising the ambient temperature from 22 to 37 degrees C, suggesting a thermally activated process. Surprisingly, SNAP-25 had little effect on docking efficiency or the probability of thermally induced fusion. Interprotein fluorescence resonance energy transfer experiments suggest the presence of other conformational states of the syntaxin*synaptobrevin interaction in addition to those observed in the crystal structure of the SNARE complex. Furthermore, although SNARE complexes involved in liposome docking preferentially assemble into a parallel configuration, both parallel and antiparallel configurations were observed.