Characterization of the Walker A motif of MsbA using site-directed spin labeling electron paramagnetic resonance spectroscopy

MsbA is an ABC transporter that transports lipid A across the inner membrane of Gram-negative bacteria such as Escherichia coli. Without functional MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, several functionally important motifs common to ABC transporters are unresolved in the crystal structure. The Walker A domain, one of the ABC transporter consensus motifs that is directly involved in ATP binding, is located within a large unresolved region of the MsbA ATPase domain. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions. MsbA reconstituted into lipid membranes has been evaluated by EPR spectroscopy, and it has been determined that the Walker A domain forms an alpha-helical structure, which is consistent with the structure of this motif observed in other crystallized ABC transporters. In addition, the interaction of the Walker A residues with ATP before, during, and after hydrolysis was followed using SDSL EPR spectroscopy in order to identify the residues directly involved in substrate binding and hydrolysis.     

Electron spin relaxation of the electron paramagnetic resonance spectra of cytochrome c

The progressive power saturation of the electron paramagnetic resonance (EPR) spectrum of ferricytochrome c has been investigated in order to determine the spin-lattice relaxation time of the center. We have generalized the usual saturation treatments to include the effects of extended sample size and anisotropic g values as well as derivative spectra. We find that the results are consistent with a T7 power law in the temperature range 6--25 K. At temperatures above 25 K the relaxation time is too short for successful power saturation. Observation of the linewidth shows that the relaxation behavior continues as a first-order Raman process to 50 K.     

Characterization of structural changes in vimentin bearing an epidermolysis bullosa simplex-like mutation using site-directed spin labeling and electron paramagnetic resonance

Mutations in intermediate filament protein genes are responsible for a number of inherited genetic diseases including skin blistering diseases, corneal opacities, and neurological degenerations. Mutation of the arginine (Arg) residue of the highly conserved LNDR motif has been shown to be causative in inherited disorders in at least four different intermediate filament (IF) proteins found in skin, cornea, and the central nervous system. Thus this residue appears to be broadly important to IF assembly and/or function. While the genetic basis for these diseases has been clearly defined, the inability to determine crystal structure for IFs has precluded a determination of how these mutations affect assembly/structure/function of IFs. To investigate the impact of mutation at this site in IFs, we have mutated the LNDR to LNDS in vimentin, a Type III intermediate filament protein, and have examined the impact of this change on assembly using electron paramagnetic resonance. Compared with wild type vimentin, the mutant shows normal formation of the coiled coil dimer, with a slight reduction in the stability of the dimer in rod domain 1. Probing the dimer-dimer interactions shows the formation of normal dimer centered on residue 191 but a failure of dimerization at residue 348 in rod domain 2. These data point toward a specific stage of assembly at which a common disease-causing mutation in IF proteins interrupts assembly.     

High-field electron spin resonance of spin labels in membranes

High-field electron spin resonance (ESR) spectroscopy is currently undergoing rapid development. This considerably increases the versatility of spin labelling which, at conventional field strengths, is already well established as a powerful physical technique in membrane biology. Among the unique advantages offered by high-field spectroscopy, particularly for spin-labelled lipids, are sensitivity to non-axial rotation and lateral ordering, a better orientational selection, an extended application to rotational dynamics, and an enhanced sensitivity to environmental polarity. These areas are treated in some depth, along with a detailed consideration of recent developments in the investigation of transmembrane polarity profiles.     

Electron paramagnetic resonance study of structural changes in the O photointermediate of bacteriorhodopsin

The structural changes of bacteriorhodopsin during its photochemical cycle, as revealed by crystal structures of trapped intermediates, have provided insights to the proton translocation mechanism. Because accumulation of the last photointermediate, O, appears to be hindered by lattice forces in the crystals, the only information about the structure of this state is from suggested analogies with the determined structures of the non-illuminated D85S mutant and wild-type bacteriorhodopsin at low pH. We used electron paramagnetic resonance spectroscopy of site-directed spin labels at the extracellular protein surface in membranes to test these models. Spin-spin dipolar interactions in the authentic O state compared to the non-illuminated state revealed that the distance between helices C and F increases by ca 4 Angstroms, there is no distance change between helices D and F, and the distance between helix D and helix B of the adjacent monomer increases. Further, the mobility changes of single labels indicate that helices E and F move outward from the proton channel at the center of the protein, and helix D tilts inward. The overall pattern of movements suggests that the model at acid pH is a better representation of the O state than D85S. However, the mobility analysis of spin-labels on the B-C interhelical loop indicates that the antiparallel beta-sheet maintains its ordered secondary structure in O, instead of the predicted disorder in the two structural models. During decay of the O state, the last step of the photocycle, a proton is transferred from Asp85 to proton release complex in the extracellular proton channel. The structural changes in O suggest the need of large conformational changes to drive the Arg82 side-chain back to its initial orientation towards Asp85, and to rearrange the numerous water molecules in this region in order to conduct the proton away from Asp85.     

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide

Nitric oxide (NO) is a free radical species with multiple physiological functions. Because of low concentrations and short half-life of NO, its direct measurement in living tissues remains a difficult task. Electron paramagnetic resonance (EPR) spin trapping is probably one of the best suitable platforms for development of new methods for quantification of biological NO. The most reliable EPR-based approaches developed so far are based on the reaction of NO with various iron complexes, both intrinsic and exogenously applied. This review is focused on the current state and perspectives of EPR spin trapping for experimental and clinical NO biology.     

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.     

Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance

Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.     

Identification of phosphorylation-induced changes in vimentin intermediate filaments by site-directed spin labeling and electron paramagnetic resonance

Phosphorylation drives the disassembly of the vimentin intermediate filament (IF) cytoskeleton at mitosis. Chromatographic analysis has suggested that phosphorylation produces a soluble vimentin tetramer, but little has been determined about the structural changes that are caused by phosphorylation or the structure of the resulting tetramer. In this study, site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) were used to examine the structural changes resulting from protein kinase A phosphorylation of vimentin IFs in vitro. EPR spectra suggest that the tetrameric species resulting from phosphorylation is the A11 configuration. EPR spectra also establish that the greatest degree of structural change was found in the linker 2 and the C-terminal half of the rod domain, despite the fact that most phosphorylation occurs in the N-terminal head domain. The phosphorylation-induced changes notably affected the proposed "trigger sequences" located in the linker 2 region, which have been hypothesized to mediate the induction of coiled-coil formation. These data are the first to document specific changes in IF structure resulting from a physiologic regulatory mechanism and provide further evidence, also generated by SDSL-EPR, that the linker regions play a key role in IF structure and regulation of assembly/disassembly.     

Electron spin labeling reveals the highly dynamic N-terminal arms of the SOS mutagenesis protein UmuD

Electron paramagnetic resonance (EPR) spectroscopy was used to probe the conformational dynamics of the N-terminal arms of the umuD gene products. We determined that the arms of UmuD(2) display a large degree of motion, are largely unbound from the globular C-terminal domain, and that the free energy of dissociation is +2.1 kJ mol(-1).     

Selective placement of electron spin resonance spin labels: new structural methods for peptides and proteins.

Electron spin resonance (ESR) is more powerful than ever as a technique for solving biochemical and biophysical problems. Part of the great utility of ESR arises from the use of modern biochemical methods to place spin labels at important positions along the primary sequence of a peptide or protein.     

Electron paramagnetic resonance study reveals a putative iron-sulfur cluster in human rpS3 protein

The human ribosomal protein S3 (rpS3) functions as a component of the 40S subunit and as a UV DNA repair endonuclease. This enzyme has an endonuclease activity for UV-irradiated and oxidatively damaged DNAs. DNA repair endonucleases recognize a variety of UV and oxidative base damages in DNA from E. coli to human cells. E. coli endonuclease III is especially known to have an iron-sulfur cluster as a co-factor. Here, we tried an electron paramagnetic resonance (EPR) method for the first time to observe a known iron-sulfur cluster signal from E. coli endonuclease III that was previously reported. We compared it to the human rpS3 in order to find out whether or not the human protein contains an iron-sulfur cluster. As a result, we succeeded in observing a Fe EPR signal that is apparently from an iron-sulfur cluster in the human rpS3 endonuclease. The EPR signal from the human enzyme, consisting of three major parts, is similar to that from the E. coli enzyme, but it has a distinct extra peak.     

Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Electron paramagnetic resonance studies of a ras p21-MnIIGDP complex in solution.

The number of water molecules bound to Mn2+ in the complex with a variant of Ha ras p21 and GDP has been determined by electron paramagnetic resonance (EPR) measurements in 17O-enriched water. A resolution enhancement method has been used to improve quantitation of the spectral data. These spectroscopic measurements show that Mn2+ has four water ligands in this complex, a result in agreement with the conclusions of a previous paper [Smithers, G. W., Poe, M., Latwesen, D. G., & Reed, G. H. (1990) Arch. Biochem. Biophys. 280, 416-420]. The resolution enhancement method has also been applied in a measurement of the 17O-Mn2+ superhyperfine coupling constant of 17O in the beta-phosphate of the GDP in the ras p21 complex. The intrinsically narrow EPR signals of Mn2+ in the complex with ras p21 and GDP in 2H2O respond to resolution enhancement such that the superhyperfine splitting from the 17O nuclear spin (I = 5/2) becomes visible in the EPR signals. An 17O-Mn2+ superhyperfine coupling constant is obtained from simulation of the resolution-enhanced EPR spectrum.     

Manganese as a calcium probe: Electron paramagnetic resonance and nuclear magnetic resonance spectroscopy of intact cells

Summary When Lettree cells are exposed to Mn²⁺, the cation becomes associated with cells in two ways: in a relatively loose and mobile manner that gives a six-line EPR spectrum designated Mn _b ^*, and in an immobile, relatively tight manner that gives no detectable EPR spectrum, designated Mn _b . Mn _b ^* is probably on the surface of cells; most Mn _b is probably inside cells. NMR measurements of Lettree cell suspensions show two water proton relaxation rates and confirm the existence of cell-associated Mn. Human erythrocytes, on the other hand, bind no Mn²⁺ under these conditions, as judged by EPR and NMR measurements.Virally-treated Lettree cells show an increase in Mn _b (but not in Mn _b ^*). They also show a third water proton relaxation rate.     

Electron paramagnetic resonance characterization of tyrosine radical, M+, in site-directed mutants of photosystem II(t).

Photosystem II contains two well-characterized tyrosine radicals, D(.) and Z(.). Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site and is essential for enzymatic function. On the other hand, D forms a stable radical with no known role in oxygen evolution. D(.) and Z(.) give rise to similar, but not identical, room temperature electron paramagnetic resonance (EPR) signals, which can be distinguished by their decay kinetics. A third room temperature EPR signal has also been observed in site-directed mutants in which a nonredox active amino acid is substituted at the D or Z site. This four-line EPR signal has been shown to have a tyrosine origin by isotopic labeling (Boerner and Barry, 1994, J. Biol. Chem. 269:134-137), but such an EPR signal has never before been observed from a tyrosyl radical. The radical giving rise to this third unique signal has been named M+. Here we provide kinetic evidence that this signal arises from a third redox active tyrosine, distinct from tyrosine D and Z, in the photosystem II reaction center. Isotopic labeling and EPR spectroscopy provide evidence that M is a covalently modified tyrosine.     

Formate dehydrogenase from Methanobacterium formicicum. Electron paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers

Formate dehydrogenase from Methanobacterium formicicum was examined by electron paramagnetic resonance spectroscopy. Although oxidized enzyme yielded no EPR signals over the temperature range 8-200 K, dithionite reduction resulted in generation of two paramagnetic components. The first, a nearly isotropic signal visible at temperatures below 200 K with g1 = 2.018, g2 = 2.003, and g3 = 1.994, exhibited nuclear hyperfine interaction with two equivalent protons (A1 = 0.45, A2 = 0.6, and A3 = 0.55 milliTeslas). EPR spectra of partially reduced 95Mo-enriched formate dehydrogenase exhibited additional 3-4 milliTeslas splittings, due to spin interaction with the 95Mo nucleus. Thus, this signal is due to a Mo center. This is the first reported example of a Mo center with gav greater than 2.0 in a biological system. The second species, a rhombic signal visible below 40 K with g values of g1 = 2.0465, g2 = 1.9482, and g3 = 1.9111 showed no hyperfine coupling and was assigned to reduced Fe/S. Both paramagnetic species could be detected in samples of M. formicicum whole cells anaerobically reduced with sodium formate. The Mo(V) signal was altered following addition of cyanide (g1 = 1.996, g2 = 1.988, and g3 = 1.980). Growth of bacteria in the presence of 1 mM WO4(2-) resulted in abolition of the Mo(V) EPR signal and formate dehydrogenase activity. Em, 7.7 was -330 mV for Mo(VI)/Mo(V) and -470 mV for Mo(V)/Mo(IV).