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1.
Biophys J ; 81(6): 3156-65, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11720982

ABSTRACT

Computational methods have been developed to model the effects of constrained or restricted amplitude uniaxial rotational diffusion (URD) on saturation transfer electron paramagnetic resonance (ST-EPR) signals observed from nitroxide spin labels. These methods, which have been developed to model the global rotational motion of intrinsic membrane proteins that can interact with the cytoskeleton or other peripheral proteins, are an extension of previous work that described computationally efficient algorithms for calculating ST-EPR spectra for unconstrained URD (Hustedt and Beth, 1995, Biophys. J. 69:1409-1423). Calculations are presented that demonstrate the dependence of the ST-EPR signal (V'(2)) on the width (Delta) of a square-well potential as a function of the microwave frequency, the correlation time for URD, and the orientation of the spin-label with respect to the URD axis. At a correlation time of 10 micros, the V'(2) signal is very sensitive to Delta in the range from 0 to 60 degrees, marginally sensitive from 60 degrees to 90 degrees, and insensitive beyond 90 degrees. Sensitivity to Delta depends on the correlation time for URD with higher sensitivity to large values of Delta at the shorter correlation times, on the microwave frequency, and on the orientation of the spin-label relative to the URD axis. The computational algorithm has been incorporated into a global nonlinear least-squares analysis approach, based upon the Marquardt-Levenberg method (Blackman et al., 2001, Biophys. J. 81:3363-3376). This has permitted determination of the correlation time for URD and the width of the square-well potential by automated fitting of experimental ST-EPR data sets obtained from a spin-labeled membrane protein and provided a new automated method for analysis of data obtained from any system that exhibits restricted amplitude URD.


Subject(s)
Diffusion , Electron Spin Resonance Spectroscopy/methods , Algorithms , Least-Squares Analysis , Microwaves , Nitrogen Oxides/chemistry , Software
2.
Biophys J ; 81(6): 3363-76, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11720999

ABSTRACT

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.


Subject(s)
Anion Exchange Protein 1, Erythrocyte/chemistry , Cytoplasm/chemistry , Erythrocytes/metabolism , Anisotropy , Cell Membrane/metabolism , Cytoplasm/metabolism , Humans , Models, Chemical , Protein Structure, Tertiary , Surface Plasmon Resonance , Time Factors , Trypsin/chemistry , Trypsin/pharmacology
3.
Article in English | MEDLINE | ID: mdl-10410798

ABSTRACT

Measurement of the distance between two spin label probes in proteins permits the spatial orientation of elements of defined secondary structure. By using site-directed spin labeling, it is possible to determine multiple distance constraints and thereby build tertiary and quaternary structural models as well as measure the kinetics of structural changes. New analytical methods for determining interprobe distances and relative orientations for uniquely oriented spin labels have been developed using global analysis of multifrequency electron paramagnetic resonance data. New methods have also been developed for determining interprobe distances for randomly oriented spin labels. These methods are being applied to a wide range of structural problems, including peptides, soluble proteins, and membrane proteins, that are not readily characterized by other structural techniques.


Subject(s)
Cyclic N-Oxides , Peptides/chemistry , Protein Conformation , Proteins/chemistry , Spin Labels , Electron Spin Resonance Spectroscopy/methods , Membrane Proteins/chemistry , Protein Structure, Tertiary
4.
Biophys J ; 75(2): 1117-30, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9675213

ABSTRACT

The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric and/or tetrameric band 3, and does not require postulation of a fraction of large clusters. Proteolytic removal of the cytoplasmic domain of band 3, which significantly increases the rotational mobility of the transmembrane domain, does not affect its oligomeric state, as reported by eosin homotransfer. These results support a model in which interaction with the membrane skeleton restricts the mobility of band 3 without significantly altering its self-association state.


Subject(s)
Anion Exchange Protein 1, Erythrocyte/chemistry , Erythrocyte Membrane/physiology , Anion Exchange Protein 1, Erythrocyte/isolation & purification , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Dimerization , Electrophoresis, Polyacrylamide Gel , Eosine Yellowish-(YS)/analogs & derivatives , Erythrocyte Membrane/drug effects , Humans , Lysine , Macromolecular Substances , Melitten/pharmacology , Models, Chemical , Spectrometry, Fluorescence/methods , Spectroscopy, Fourier Transform Infrared/methods , Zinc/pharmacology
5.
Biophys J ; 72(4): 1861-77, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9083690

ABSTRACT

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar splittings can be observed in continuous wave electron paramagnetic resonance (CW-EPR) spectra for interelectron distances as large as 30 A using perdeuterated probes. In this work, algorithms are developed for calculating CW-EPR spectra of immobilized, dipolar coupled nitroxides, and then used to define the limits of sensitivity to the interelectron distance as a function of geometry and microwave frequency. Secondly, the CW-EPR spectra of N epsilon-spin-labeled coenzyme NAD+ bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of simulated annealing and global analysis, to obtain a unique fit to the data. The values of the intermitroxide distance and the five angles defining the relative orientation of the two nitroxides are in reasonable agreement with a molecular model built from the known crystal structure. Finally, the effect of rigid body isotropic rotational diffusion on the CW-EPR spectra of dipolar coupled nitroxides has been investigated using an algorithm based on Brownian dynamics trajectories. These calculations demonstrate the sensitivity of CW-EPR spectra to dipolar coupling in the presence of rigid body rotational diffusion.


Subject(s)
Electron Spin Resonance Spectroscopy , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , NAD/metabolism , Spin Labels , Algorithms , Animals , Diffusion , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Mathematics , Molecular Structure , Muscle, Skeletal/enzymology , Protein Conformation , Rabbits
6.
Biophys J ; 71(1): 194-208, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8804603

ABSTRACT

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.


Subject(s)
Anion Exchange Protein 1, Erythrocyte/chemistry , Eosine Yellowish-(YS)/analogs & derivatives , Fluorescent Dyes/chemistry , Biophysical Phenomena , Biophysics , Diffusion , Electron Spin Resonance Spectroscopy , Eosine Yellowish-(YS)/chemistry , Erythrocyte Membrane/chemistry , Fluorescence Polarization , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Models, Chemical , Rotation , Thermodynamics , Trypsin
7.
Biochemistry ; 35(21): 6931-43, 1996 May 28.
Article in English | MEDLINE | ID: mdl-8639645

ABSTRACT

A new spin-labeled maleimide derivative of the anion exchange inhibitor 4-4'-diaminodihydrostilbene-2,2'-disulfonate (H2DADS) has been synthesized as a site-specific molecular probe of the stilbenedisulfonate binding site of the anion exchange protein 1 (AE-1; band 3) in human erythrocytes. This probe, SL-H2DADS-maleimide, specifically and covalently labels the Mr 17 kDa integral membrane segment of band 3 with a 1:1 stoichiometry and inhibits essentially 100% of the band 3-mediated anion exchange. The linear V1 EPR spectrum of spin-labeled intact erythrocytes is indicative of a spatially isolated probe which is effectively immobilized on the submicrosecond time scale. Several independent lines of experimental evidence have shown that the nitroxide moiety of SL-H2DADS-maleimide-labeled band 3 is sequestered in a highly protected protein environment. These results are consistent with the observation that the spin-label is rigidly linked to band 3 in a fixed orientation with respect to the membrane normal axis [Hustedt, E. J., & Beth, A. H., (1996) Biochemistry 35, 6944-6954]. The nitroxide moieties of the SL-H2DADS-maleimide-labeled band 3 dimer are greater than 20 A from each other and are also more than 20 A from a monomer-monomer contact surface defined by cross-linking with the spin-labeled reagent BSSDA [bis(sulfo-N-succinimidyl)doxyl-2-spiro-5'-azelate]. These properties make SL-H2DADS-maleimide an extremely useful molecular probe for characterization of the physical properties of the band 3 stilbenedisulfonate binding site, determination of distances between the stilbenedisulfonate site and other segments of band 3, and investigation of the global rotational dynamics of human erythrocyte band 3.


Subject(s)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , Affinity Labels/chemical synthesis , Anion Exchange Protein 1, Erythrocyte/chemistry , Erythrocyte Membrane/metabolism , Spin Labels/chemical synthesis , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/chemical synthesis , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/chemistry , Anion Exchange Protein 1, Erythrocyte/isolation & purification , Anion Exchange Protein 1, Erythrocyte/metabolism , Biological Transport , Electron Spin Resonance Spectroscopy/methods , Erythrocyte Membrane/ultrastructure , Humans , Indicators and Reagents , Sulfates/blood
8.
Biochemistry ; 35(21): 6944-54, 1996 May 28.
Article in English | MEDLINE | ID: mdl-8639646

ABSTRACT

The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.


Subject(s)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , Anion Exchange Protein 1, Erythrocyte/chemistry , Erythrocyte Membrane/ultrastructure , Spin Labels , Anion Exchange Protein 1, Erythrocyte/metabolism , Deuterium , Electron Spin Resonance Spectroscopy/methods , Erythrocyte Membrane/metabolism , Humans , Kinetics , Least-Squares Analysis , Mathematics , Models, Theoretical , Nitrogen Isotopes , Radioisotope Dilution Technique
9.
Biophys J ; 69(4): 1409-23, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8534811

ABSTRACT

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer.


Subject(s)
Anion Exchange Protein 1, Erythrocyte/chemistry , Models, Theoretical , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , Algorithms , Diffusion , Electron Spin Resonance Spectroscopy/methods , Erythrocyte Membrane/physiology , Humans , Rotation , Spin Labels
10.
FEBS Lett ; 367(2): 137-40, 1995 Jun 26.
Article in English | MEDLINE | ID: mdl-7540989

ABSTRACT

The separations between aromatic residues in the bait region and nitroxide spin labels attached to the thiol ester-forming residues (Cys949 and Gln952) in human alpha 2-macroglobulin (alpha 2M) have been determined from paramagnetic broadening effects of the spin labels on bait region 1H NMR signals. We found that both the Cys949 and Gln952 residues are within 11-17 A of the aromatic residues in the bait region, with closer approach of some residues to the Gln952 spin label than to the spin label attached to Cys949. A model of the location of bait regions and thiol esters within an alpha 2M half-molecule is proposed that places the bait regions in the central region of alpha 2M at the interface between the subunits.


Subject(s)
alpha-Macroglobulins/chemistry , Electron Spin Resonance Spectroscopy , Esters , Humans , Magnetic Resonance Spectroscopy , Spin Labels , Sulfhydryl Compounds
11.
Biochemistry ; 32(36): 9454-64, 1993 Sep 14.
Article in English | MEDLINE | ID: mdl-8396432

ABSTRACT

Two new spin-label derivatives of 4,4'-diaminodihydrostilbene-2,2'-disulfonate (H2-DADS) have been chemically synthesized and employed in electron paramagnetic resonance (EPR) studies of binding to the anion exchange protein (band 3) in intact human erythrocytes. Equilibrium binding studies with the 4-monoacyl-spin-label derivative (mono-SL-H2-DADS) indicated an effective dissociation constant of 11 microM and substantial negative cooperativity in isotonic citrate buffer, pH 7.4, at 20 degrees C. The 4,4'-diacyl-spin-label derivative (di-SL-H2-DADS) bound with an effective dissociation constant of 54 microM and no detectable cooperativity under the same binding conditions. The findings of substantial negative cooperativity in binding of the less bulky mono-SL-H2-DADS and no cooperativity for di-SL-H2-DADS suggest the presence of an allosteric coupling between the stilbenedisulfonate sites on adjacent band 3 monomers rather than steric interactions. There were approximately 1 x 10(6) binding sites per erythrocyte for both the mono- and di-SL-H2-DADS derivatives, and the binding of each was blocked by pretreatment of intact cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a highly specific covalent inhibitor of anion exchange. EPR spectra collected over a wide range of concentrations of mono-SL-H2-DADS indicated that binding resulted in immobilization of the probe and that, even upon near saturation of available binding sites, there were no detectable dipole-dipole interactions between bound probes. EPR spectra collected using di-SL-H2-DADS revealed the presence of intramolecular dipole-dipole interactions between spin-label moieties on opposite ends of this biradical probe, but no intermolecular dipole-dipole interactions between separate bound probes. These data indicate that di-SL-H2-DADS binds to the stilbenedisulfonate binding site on band 3 in a bent conformation and further suggest that the termini of these binding sites on adjacent monomers are greater than 20 A apart.


Subject(s)
Anion Exchange Protein 1, Erythrocyte/metabolism , Erythrocytes/metabolism , Stilbenes/metabolism , Binding Sites , Cells, Cultured , Electron Spin Resonance Spectroscopy , Electrons , Humans , Models, Molecular , Spin Labels
12.
Biochemistry ; 32(31): 7893-903, 1993 Aug 10.
Article in English | MEDLINE | ID: mdl-8394119

ABSTRACT

In this report we describe the production of a [Lys3,Tyr22] murine epidermal growth factor (mEGF) mutant for spin-labeling with bis(sulfo-N-succinimidyl)-[15N,2H16]doxyl-2-spiro-4'-pimelate ([15N,2H16]BSSDP) in order to study the rotational dynamics of the EGF/EGF receptor complex by saturation-transfer electron paramagnetic resonance (ST-EPR). Previous results [Faulkner-O'Brien et al. (1991) Biochemistry 30,8976-8985] indicated that the reaction of [15N,2H16]BSSDP with wild-type mEGF did not yield a product useful for ST-EPR studies of the EGF/EGF receptor complex because the major product, in which [15N,2H16]BSSDP was attached only at the amino terminus of mEGF, lacked rigid motional coupling of the spin probe to the protein, and the more tightly coupled bidentate product was unstable. Using oligonucleotide-mediated site-directed mutagenesis of a synthetic gene for mEGF, we replaced Tyr3 with Lys and His22 with Tyr in wild-type mEGF to produce a mutant mEGF suitable for [15N,2H16]BSSDP labeling. The [Lys3,Tyr22] mEGF was expressed in Escherichia coli HB101 transformed with a pIN-III-ompA3-[Lys3,Tyr22] mEGF plasmid and was purified from the bacterial periplasm using a simple two step purification method. The [15N,2H16]BSSDP reacted with [Lys3,Tyr22]mEGF in high yield, and EPR analysis of the major product revealed tight motional coupling between the spin probe and the protein. Biological activity, as assessed by stimulation of EGF receptor autophosphorylation and dimerization, was not affected by either the mutations or the addition of the spin label. The [15N,2H16]BSSDP-modified [Lys3,Tyr22] mEGF was shown to be equipotent with mEGF in EGF receptor competition binding assays using A431 cells; in EPR studies, mEGF also was shown to specifically block [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF binding to the EGF receptor in A431 membrane vesicles. Using the [15N,2H16]BSSDP-modified [Lys3,Tyr22]mEGF, we now report the first measurement of the rotational dynamics of the EGF/EGF receptor complex in A431 membrane vesicles by ST-EPR.


Subject(s)
Epidermal Growth Factor/chemistry , ErbB Receptors/chemistry , Amino Acid Sequence , Base Sequence , Electron Spin Resonance Spectroscopy , Epidermal Growth Factor/biosynthesis , Escherichia coli , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Spin Labels , Succinimides , Tumor Cells, Cultured
13.
Biophys J ; 64(3): 605-13, 1993 Mar.
Article in English | MEDLINE | ID: mdl-7682451

ABSTRACT

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.


Subject(s)
Fluorescent Dyes , Proteins/chemistry , Spin Labels , Biophysical Phenomena , Biophysics , Electron Spin Resonance Spectroscopy , Eosine Yellowish-(YS)/analogs & derivatives , Eosine Yellowish-(YS)/chemical synthesis , Fluorescence Polarization , Fluorescent Dyes/chemical synthesis , Luminescence , Molecular Probes , Motion , Rotation , Serum Albumin, Bovine/chemistry , Solutions , Spin Labels/chemical synthesis
14.
Biophys J ; 64(3): 614-21, 1993 Mar.
Article in English | MEDLINE | ID: mdl-7682452

ABSTRACT

In the preceding companion article in this issue, an optical dye and a nitroxide radical were combined in a new dual function probe, 5-SLE. In this report, it is demonstrated that time-resolved optical anisotropy and electron paramagnetic resonance (EPR) data can be combined in a single analysis to measure rotational dynamics. Rigid-limit and rotational diffusion models for simulating nitroxide EPR data have been incorporated into a general non-linear least-squares procedure based on the Marquardt-Levenberg algorithm. Simultaneous fits to simulated time-resolved fluorescence anisotropy and linear EPR data, together with simultaneous fits to experimental time-resolved phosphorescence anisotropy decays and saturation transfer EPR (ST-EPR) spectra of 5-SLE noncovalently bound to bovine serum albumin (BSA) have been performed. These results demonstrate that data from optical and EPR experiments can be combined and globally fit to a single dynamic model.


Subject(s)
Electron Spin Resonance Spectroscopy , Proteins/chemistry , Biophysical Phenomena , Biophysics , Data Interpretation, Statistical , Diffusion , Electron Spin Resonance Spectroscopy/statistics & numerical data , Eosine Yellowish-(YS)/analogs & derivatives , Fluorescence Polarization/statistics & numerical data , Fluorescent Dyes , Models, Chemical , Motion , Rotation , Serum Albumin, Bovine/chemistry , Spin Labels
15.
Biochemistry ; 30(37): 8976-85, 1991 Sep 17.
Article in English | MEDLINE | ID: mdl-1654082

ABSTRACT

We prepared, purified, and characterized derivatives of epidermal growth factor (EGF) having a nitroxide spin-label attached covalently at the amino terminus. Characterization of these derivatives with regard to the positions of attachment of the spin-label was accomplished by a combination of peptide mapping, protein sequencing, and fast atom bombardment-mass spectrometry. One derivative was chosen for use in initial investigations by electron paramagnetic resonance (EPR) spectroscopy of receptor-bound EGF and its dissociation kinetics. This derivative was found to be equipotent with the native hormone in competitive binding assays, in activating the EGF receptor kinase, and in stimulating the formation of EGF receptor dimers in solubilized cell extracts. Upon binding to solubilized EGF receptor, the spin-labeled EGF derivative became immobilized, giving rise to a visually distinct slow-motion EPR spectrum. The resulting spectrum showed no detectable dipolar interaction between nitroxides, indicating that the nitroxide moieties of spin-labels reacted at the amino termini of receptor-bound spin-labeled EGF molecules are separated by a distance of at least 16 A. An EPR study of the kinetics of dissociation of spin-labeled EGF in the presence of excess unlabeled EGF revealed a rapid component with a k off approximately 2 x 10(-2) s-1 and a less well resolved slow component.


Subject(s)
Electron Spin Resonance Spectroscopy , Epidermal Growth Factor/chemistry , ErbB Receptors/chemistry , Amino Acid Sequence , Animals , Binding, Competitive , Epidermal Growth Factor/isolation & purification , Kinetics , Mice , Molecular Sequence Data , Spectrometry, Mass, Fast Atom Bombardment , Spin Labels , Succinimides
16.
J Membr Biol ; 121(3): 261-8, 1991 May.
Article in English | MEDLINE | ID: mdl-1865490

ABSTRACT

Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.


Subject(s)
Adipose Tissue/metabolism , Fatty Acids/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/metabolism , Animals , Biological Transport, Active , In Vitro Techniques , Male , Membrane Proteins/metabolism , Rats , Rats, Inbred Strains , Succinimides/metabolism
17.
Biochemistry ; 29(48): 10799-806, 1990 Dec 04.
Article in English | MEDLINE | ID: mdl-2176884

ABSTRACT

The roles of lipid ordering and protein dynamics on the function of the anion exchange protein (band 3) in intact human erythrocytes have been investigated. The effects of diethyl ether on the ordering of membrane lipids and on the rotational dynamics of band 3 were measured by EPR and saturation-transfer EPR spectroscopies, respectively, and correlated with the anion exchange function of band 3. With increasing concentration, diethyl ether monotonically decreased the ordering of membrane lipids near the polar head-group region, as reported by the lipid-soluble spin probe 5-doxylstearic acid, but produced comparatively little change in the ordering of lipids in the hydrophobic midzone, as reported by 16-doxylstearic acid. The rotational mobility of band 3, as reported by the affinity spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate [Anjaneyulu et al. (1989) Biochemistry 28, 6583-6590], also increased monotonically with increasing ether concentration. This increase in rotational mobility was not due to a demonstrable change in its state of oligomerization, since band 3 was readily cross-linked by bis(sulfo-N-succinimidyl) suberate to covalent dimers in the presence or absence of ether. At concentrations up to 2 vol % ether, hemolysis of erythrocytes was negligible, and the spectroscopic changes observed were completely reversed following its removal. Km, Vmax, and Eact. for sulfate uptake into chloride-loaded erythrocytes were not significantly affected by addition of ether.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Carrier Proteins/blood , Erythrocyte Membrane/physiology , Erythrocytes/physiology , Ether/pharmacology , Membrane Lipids/blood , Anion Exchange Protein 1, Erythrocyte/chemistry , Anion Transport Proteins , Carrier Proteins/chemistry , Chemical Phenomena , Chemistry, Physical , Chlorides/blood , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , Erythrocytes/chemistry , Erythrocytes/drug effects , Humans , Kinetics , Macromolecular Substances , Membrane Fluidity , Membrane Lipids/chemistry , Spin Labels , Sulfates/blood , Thermodynamics
18.
Magn Reson Med ; 15(3): 397-419, 1990 Sep.
Article in English | MEDLINE | ID: mdl-2233220

ABSTRACT

A pulse sequence is presented for obtaining a single image with combined T1/T2 weighting. T2 relaxation is made to increase intensity, in cooperation with the effect of T1 relaxation, by providing T2 weighting with a 90 degrees-180 degrees-90 degrees driven inversion pulse triplet in an inversion recovery method. Unlike the inversion spin-echo method having a short inversion time (TI), signals in the new driven inversion spin-echo (DISE) method need not be negative and the most T1-sensitive region of the recovery curve can be used. Selecting sensitivity to one relaxation time does not degrade the sensitivity to the other relaxation time. T1 sensitivity is thus extended to longer echo times (TE intervals). T2 sensitivity is extended to longer TI intervals, and the combined T1/T2-weighted technique with intermediate TE and TI has highly cooperative and near-maximal T1 and T2 effects on contrast. Intensity is not multiplicatively degraded by T1 and T2 weighting so that the signal-to-noise of the combined T1/T2-weighted method is high. High intensity and T1 and T2 cooperatively occur for a much wider range of relaxation times, and especially for images heavily weighted to the pathologic intermediate and long T1 and T2 regime.


Subject(s)
Brain/anatomy & histology , Magnetic Resonance Imaging/methods , Humans , Image Enhancement/methods , Models, Theoretical
19.
Biochemistry ; 29(36): 8283-90, 1990 Sep 11.
Article in English | MEDLINE | ID: mdl-1701324

ABSTRACT

The anion-exchange protein (band 3) reaction site in human erythrocytes for the fluorescent/phosphorescent probe eosinyl-5-maleimide (EMA) has been identified. Proteolytic dissection of band 3 in situ indicated that EMA reacts with the membrane-spanning Mr 17K peptide produced by chymotrypsin cleavage of band 3 in intact erythrocytes followed by removal of the cytoplasmic domain by mild trypsin digestion of ghost membranes. Sequencing of the major eosin-labeled peptide obtained from HPLC purification of an extensive chymotrypsin digest of purified Mr 17K peptide allowed assignment of the covalent reaction site for EMA to lysine-430 of the human erythrocyte protein [Tanner et al. (1988) Biochem. J. 256, 703-712]. Hydropathy plots based upon the primary structure of the protein [Lux et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9089-9093] suggest that this residue is in an extracellularly accessible loop connecting membrane-spanning segments 1 and 2 of native band 3 in the erythrocyte membrane. Inhibition of sequential labeling of intact erythrocytes by pairs of chemical probes including EMA, the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2-DIDS), and the reactively bifunctional spin-label bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA) has also been investigated. Each of these reagents affinity labels band 3 when added separately to a suspension of intact human erythrocytes by formation of one or more stable covalent bonds. Prelabeling of intact erythrocytes with EMA reduced subsequent labeling of band 3 by H2-DIDS by approximately 95% and by BSSDA by 90%. Similarly, prelabeling with H2-DIDS reduced subsequent labeling of band 3 by EMA by over 90%, and BSSDA prelabeling reduced EMA labeling by approximately 95%. Therefore, though having widely divergent chemical structures and protein modification reactivities, each of these negatively charged reagents may be competing for reaction with spatially overlapping sites on band 3 which are accessible from the extracellular space.


Subject(s)
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/analogs & derivatives , Anion Exchange Protein 1, Erythrocyte/metabolism , Carrier Proteins/metabolism , Eosine Yellowish-(YS)/metabolism , Erythrocyte Membrane/metabolism , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/analogs & derivatives , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid/metabolism , Amino Acid Sequence , Anion Exchange Protein 1, Erythrocyte/antagonists & inhibitors , Anion Transport Proteins , Binding Sites , Carrier Proteins/antagonists & inhibitors , Electron Spin Resonance Spectroscopy , Humans , Molecular Sequence Data , Oxazoles/metabolism , Spin Labels , Succinimides/metabolism
20.
Biochemistry ; 28(16): 6583-90, 1989 Aug 08.
Article in English | MEDLINE | ID: mdl-2477061

ABSTRACT

We have synthesized and characterized bis(sulfo-N-succinimidyl) doxyl-2-spiro-5'-azelate (BSSDA), a membrane-impermeant bifunctional spin-labeling reagent. BSSDA is a nine carbon backbone homologue of bis(sulfo-N-succinimidyl) doxyl-2-spiro-4'-pimelate [BSSDP; Beth et al. (1986) Biochemistry 25, 3824-3832]. Due to its longer backbone, BSSDA can span longer distances between reactive groups on a protein than can BSSDP. However, the purpose of the bifunctional design of these reagents is to provide a tight motional coupling of the spin labels to the surface of a target protein. To test whether the longer backbone of BSSDA results in a greater local flexibility and thereby undermines the effects of bidentate attachment, we have labeled with BSSDA anion-exchange channels of intact human erythrocytes at the same site as we have previously labeled them with BSSDP. Linear and saturation-transfer EPR spectra of BSSDA-labeled anion-exchange channels in intact cells closely approximate the corresponding spectra from BSSDP-labeled channels. Thus, the longer backbone of BSSDA relative to BSSDP does not give rise to significant local flexibility, even when BSSDA is bound to a site that can be spanned by the shorter reagent.


Subject(s)
Erythrocytes/metabolism , Ion Channels/metabolism , Oxazoles/chemical synthesis , Succinimides/chemical synthesis , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid , 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid , Anions , Cross-Linking Reagents/chemical synthesis , Electron Spin Resonance Spectroscopy , Humans , In Vitro Techniques , Ion Exchange , Oxazoles/metabolism , Spin Labels/chemical synthesis , Succinimides/metabolism
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