Albert Beth - Academia.edu (original) (raw)

Papers by Albert Beth

Research paper thumbnail of How to Assemble the Parts: Structures of Protein Complexes from their Components

Biophysical Journal, 2007

Research paper thumbnail of A Straightforward Approach to the Analysis of Double Electron-Electron Resonance Data

Methods in enzymology, 2015

Double electron-electron resonance (DEER) is now widely utilized to measure distance distribution... more Double electron-electron resonance (DEER) is now widely utilized to measure distance distributions in the 20-70Å range. DEER is frequently applied to biological systems that have multiple conformational states leading to complex distance distributions. These complex distributions raise issues regarding the best approach to analyze DEER data. A widely used method utilizes a priori background correction followed by Tikhonov regularization. Unfortunately, the underlying assumptions of this approach can impact the analysis. In this chapter, a method of analyzing DEER data is presented that is ideally suited to obtain these complex distance distributions. The approach allows the fitting of raw experimental data without a priori background correction as well as the rigorous determination of uncertainties for all fitting parameters. This same methodological approach can be used for the simultaneous or global analysis of multiple DEER data sets using variable ratios of a common set of compo...

Research paper thumbnail of Enhancement of free radical reduction by elevated concentrations of ascorbic acid in avian dystrophic muscle

Proceedings of the National Academy of Sciences, 1980

It has been postulated that the degenerative process in dystrophic muscle results from increased ... more It has been postulated that the degenerative process in dystrophic muscle results from increased concentrations of free radicals, peroxides, or lipid hydroperoxides. Therefore, the reduction of the free radical tanol (2,2,6,6-tetramethyl-4-piperidinol-1-oxyl) by extracts of muscles of dystrophic and normal chickens was studied. Pectoral (white) and thigh (red) muscles were used. For initial rate measurements, the various muscle extracts were added to an equal volume of 0.2 mM tanol. Reaction mixtures were introduced into the EPR cavity in a standard aqueous flat cell. Rates were measured by continuously monitoring the decrease in signal amplitude of the center (MI = 0) solution tanol EPR resonance line (in-phase first harmonic absorption signal). With extracts from dystrophic white muscle, the reduction rate was 75% faster than normal, whereas in dystrophic red muscle extracts the rate was normal. This agreed with previous observations that white muscle is more severely affected tha...

Research paper thumbnail of Structural Arrangement of the Intracellular Ca2+ Binding Domains of the Cardiac Na+/Ca2+ Exchanger (NCX1.1): EFFECTS OF Ca2+ BINDING

Journal of Biological Chemistry, 2012

Research paper thumbnail of Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Journal of Biological Chemistry, 2011

Research paper thumbnail of CLC Anion Channel Regulatory Phosphorylation and Conserved Signal Transduction Domains

Biophysical Journal, 2012

Research paper thumbnail of Flexibility of the Cytoplasmic Domain of the Anion Exchange Protein, Band 3, in Human Erythrocytes

Biophysical Journal, 2001

Research paper thumbnail of Oligomeric State of Human Erythrocyte Band 3 Measured by Fluorescence Resonance Energy Homotransfer

Biophysical Journal, 1998

Research paper thumbnail of Structure and Dynamics of the Calcium Binding Domains of the Na/Ca Exchanger (NCX1.1) Determined by Site Directed Spin Labeling

Biophysical Journal, 2010

For electron paramagnetic resonance (EPR) spectroscopic studies, the TOAC spin label offers the u... more For electron paramagnetic resonance (EPR) spectroscopic studies, the TOAC spin label offers the unique advantage over other conventional labels in that it reports accurate backbone motion and peptide dynamics due to its rigid nature. This label has become extremely important in EPR studies to study membrane protein topology and their associated dynamics. Additionally, some researches have also developed spectroscopic techniques using magnetically aligned (bicelles) and mechanically aligned (glass plates) lipid samples to extract additional information directly related to structural topology with respect to the membrane. Based upon the samples orientation, other anisotropic spectral parameters can also be determined. EPR spectroscopy offers a unique solution due to the fact it has a much higher sensitivity and also a different frequency domain than other conventional techniques. Thus, we have performed EPR alignment studies on two-model peptides magainin-2 and the M2d subunit of the acetylcholine receptor. Both of these peptides have been well characterized and are 23 amino acids in length. 2060-Pos Peldor Beyond Distances Olav Schiemann. Centre for Biomolecular Sciences, St Andrews, United Kingdom. Structural Biology is engaging ever larger assemblies of biomacromolecules either isolated, embedded in membranes or in whole cells. Thus, biophysical methods are needed that access these architectures on the critical nanometer length scale in these environments. Electron Paramagnetic Resonance provides several tools to precisely and reliably measuring such these distance in the nanometer range in particular a method called Pulsed Electron-Electron Double Resonance (PELDOR). 1 In this presentation, it will be shown that PELDOR yields not only distances and distance distribution but also full information about label orientation, 2 coupling mechanisms 3 and that it can be used to count the monomers in aggregates. 4 Examples will include covalently and non-covalently labelled duplex DNAs/RNAs, complex folds of RNAs and the 320 kDa membrane channel Wza 5 .

Research paper thumbnail of Site-Directed Spin-Labeling Studies: Structure of the AnkD34-Cdb3 Complex

Biophysical Journal, 2011

Research paper thumbnail of Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data

Biophysical Journal, 1993

In the preceding companion article in this issue, an optical dye and a nitroxide radical were com... more 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 anisoptropy 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. phorescence is sensitive to motions in the range 10-6 < Tr < 100 seconds. Transient absorption anisotropy (5) and fluorescence recovery (6) are two additional methods that bridge the sensitivity ranges of the fluorescence and phosphorescence emission anisotropy methods. The use of EPR with nitroxide spin-labels to study the dynamics of lipids, DNA, proteins, and membrane bound proteins has been extensively reviewed (7, 8, 9, 10). Conventional linear EPR spectra of nitroxide spin-labels are sensitive to motions whose correlation times are in the range 10-"1 <,Tr < 10-6 seconds (11). The complementary saturation transfer EPR (ST-EPR

Research paper thumbnail of Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin

Biophysical Journal, 1993

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in ... more 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

Research paper thumbnail of Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data

Biophysical Journal, 1997

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar s... more 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 N6-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 internitroxide 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.

Research paper thumbnail of The Sensitivity of Saturation Transfer Electron Paramagnetic Resonance Spectra to Restricted Amplitude Uniaxial Rotational Diffusion

Biophysical Journal, 2001

Computational methods have been developed to model the effects of constrained or restricted ampli... more 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 (VЈ 2) on the width (⌬) 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 s, the VЈ 2 signal is very sensitive to ⌬ in the range from 0 to 60°, marginally sensitive from 60°to 90°, and insensitive beyond 90°. Sensitivity to ⌬ depends on the correlation time for URD with higher sensitivity to large values of ⌬ 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.

Research paper thumbnail of The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy

Biophysical Journal, 1996

Research paper thumbnail of Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model

Biophysical Journal, 1995

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic r... more 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 Aand 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.

Research paper thumbnail of Structure of the cdb3-ankD34 Complex from Site Directed Spin Labeling Studies

Biophysical Journal, 2009

Algorithms have been developed for calculating the continuous wave electron paramagnetic resonanc... more Algorithms have been developed for calculating the continuous wave electron paramagnetic resonance (EPR) spectrum of a nitroxide spin label from a freeinduction decay (FID) derived from a molecular dynamics (MD) simulation. These algorithms have been successfully used to simulate the EPR spectra of two spin-labeled mutants of T4 lysozyme, T4L F153R1 and T4L K65R1 310a Monday,

Research paper thumbnail of Simulation of Nitroxide Electron Paramagnetic Resonance Spectra from Brownian Trajectories and Molecular Dynamics Simulations

Biophysical Journal, 2008

Research paper thumbnail of High Field/High Frequency Saturation Transfer Electron Paramagnetic Resonance Spectroscopy: Increased Sensitivity to Very Slow Rotational Motions

Biophysical Journal, 2004

Saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy has been employed to ch... more Saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy has been employed to characterize the very slow microsecond to millisecond rotational dynamics of a wide range of nitroxide spin-labeled proteins and other macromolecules in the past three decades. The vast majority of this previous work has been carried out on spectrometers that operate at X-band (;9 GHz) microwave frequency with a few investigations reported at Q-band (;34 GHz). EPR spectrometers that operate in the 94-250-GHz range and that are capable of making conventional linear EPR measurements on small aqueous samples have now been developed. This work addresses potential advantages of utilizing these same high frequencies for ST-EPR studies that seek to quantitatively analyze the very slow rotational dynamics of spin-labeled macromolecules. For example, the uniaxial rotational diffusion (URD) model has been shown to be particularly applicable to the study of the rotational dynamics of integral membrane proteins. Computational algorithms have been employed to define the sensitivity of ST-EPR signals at 94, 140, and 250 GHz to the correlation time for URD, to the amplitude of constrained URD, and to the orientation of the spin label relative to the URD axis. The calculations presented in this work demonstrate that these higher microwave frequencies provide substantial increases in sensitivity to the correlation time for URD, to small constraints in URD, and to the geometry of the spin label relative to the URD axis as compared with measurements made at X-band. Moreover, the calculations at these higher frequencies indicate sensitivity to rotational motions in the 1-100-ms time window, particularly at 250 GHz, thereby extending the slow motion limit for ST-EPR by two orders of magnitude relative to X-and Q-bands.

Research paper thumbnail of Structure of the Cytoplasmic Domain of Erythrocyte Band 3 Hereditary Spherocytosis Variant P327R:  Band 3 Tuscaloosa †

Biochemistry, 2007

Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band ... more Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electronelectron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.

Research paper thumbnail of How to Assemble the Parts: Structures of Protein Complexes from their Components

Biophysical Journal, 2007

Research paper thumbnail of A Straightforward Approach to the Analysis of Double Electron-Electron Resonance Data

Methods in enzymology, 2015

Double electron-electron resonance (DEER) is now widely utilized to measure distance distribution... more Double electron-electron resonance (DEER) is now widely utilized to measure distance distributions in the 20-70Å range. DEER is frequently applied to biological systems that have multiple conformational states leading to complex distance distributions. These complex distributions raise issues regarding the best approach to analyze DEER data. A widely used method utilizes a priori background correction followed by Tikhonov regularization. Unfortunately, the underlying assumptions of this approach can impact the analysis. In this chapter, a method of analyzing DEER data is presented that is ideally suited to obtain these complex distance distributions. The approach allows the fitting of raw experimental data without a priori background correction as well as the rigorous determination of uncertainties for all fitting parameters. This same methodological approach can be used for the simultaneous or global analysis of multiple DEER data sets using variable ratios of a common set of compo...

Research paper thumbnail of Enhancement of free radical reduction by elevated concentrations of ascorbic acid in avian dystrophic muscle

Proceedings of the National Academy of Sciences, 1980

It has been postulated that the degenerative process in dystrophic muscle results from increased ... more It has been postulated that the degenerative process in dystrophic muscle results from increased concentrations of free radicals, peroxides, or lipid hydroperoxides. Therefore, the reduction of the free radical tanol (2,2,6,6-tetramethyl-4-piperidinol-1-oxyl) by extracts of muscles of dystrophic and normal chickens was studied. Pectoral (white) and thigh (red) muscles were used. For initial rate measurements, the various muscle extracts were added to an equal volume of 0.2 mM tanol. Reaction mixtures were introduced into the EPR cavity in a standard aqueous flat cell. Rates were measured by continuously monitoring the decrease in signal amplitude of the center (MI = 0) solution tanol EPR resonance line (in-phase first harmonic absorption signal). With extracts from dystrophic white muscle, the reduction rate was 75% faster than normal, whereas in dystrophic red muscle extracts the rate was normal. This agreed with previous observations that white muscle is more severely affected tha...

Research paper thumbnail of Structural Arrangement of the Intracellular Ca2+ Binding Domains of the Cardiac Na+/Ca2+ Exchanger (NCX1.1): EFFECTS OF Ca2+ BINDING

Journal of Biological Chemistry, 2012

Research paper thumbnail of Determination of Structural Models of the Complex between the Cytoplasmic Domain of Erythrocyte Band 3 and Ankyrin-R Repeats 13–24

Journal of Biological Chemistry, 2011

Research paper thumbnail of CLC Anion Channel Regulatory Phosphorylation and Conserved Signal Transduction Domains

Biophysical Journal, 2012

Research paper thumbnail of Flexibility of the Cytoplasmic Domain of the Anion Exchange Protein, Band 3, in Human Erythrocytes

Biophysical Journal, 2001

Research paper thumbnail of Oligomeric State of Human Erythrocyte Band 3 Measured by Fluorescence Resonance Energy Homotransfer

Biophysical Journal, 1998

Research paper thumbnail of Structure and Dynamics of the Calcium Binding Domains of the Na/Ca Exchanger (NCX1.1) Determined by Site Directed Spin Labeling

Biophysical Journal, 2010

For electron paramagnetic resonance (EPR) spectroscopic studies, the TOAC spin label offers the u... more For electron paramagnetic resonance (EPR) spectroscopic studies, the TOAC spin label offers the unique advantage over other conventional labels in that it reports accurate backbone motion and peptide dynamics due to its rigid nature. This label has become extremely important in EPR studies to study membrane protein topology and their associated dynamics. Additionally, some researches have also developed spectroscopic techniques using magnetically aligned (bicelles) and mechanically aligned (glass plates) lipid samples to extract additional information directly related to structural topology with respect to the membrane. Based upon the samples orientation, other anisotropic spectral parameters can also be determined. EPR spectroscopy offers a unique solution due to the fact it has a much higher sensitivity and also a different frequency domain than other conventional techniques. Thus, we have performed EPR alignment studies on two-model peptides magainin-2 and the M2d subunit of the acetylcholine receptor. Both of these peptides have been well characterized and are 23 amino acids in length. 2060-Pos Peldor Beyond Distances Olav Schiemann. Centre for Biomolecular Sciences, St Andrews, United Kingdom. Structural Biology is engaging ever larger assemblies of biomacromolecules either isolated, embedded in membranes or in whole cells. Thus, biophysical methods are needed that access these architectures on the critical nanometer length scale in these environments. Electron Paramagnetic Resonance provides several tools to precisely and reliably measuring such these distance in the nanometer range in particular a method called Pulsed Electron-Electron Double Resonance (PELDOR). 1 In this presentation, it will be shown that PELDOR yields not only distances and distance distribution but also full information about label orientation, 2 coupling mechanisms 3 and that it can be used to count the monomers in aggregates. 4 Examples will include covalently and non-covalently labelled duplex DNAs/RNAs, complex folds of RNAs and the 320 kDa membrane channel Wza 5 .

Research paper thumbnail of Site-Directed Spin-Labeling Studies: Structure of the AnkD34-Cdb3 Complex

Biophysical Journal, 2011

Research paper thumbnail of Measurement of rotational dynamics by the simultaneous nonlinear analysis of optical and EPR data

Biophysical Journal, 1993

In the preceding companion article in this issue, an optical dye and a nitroxide radical were com... more 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 anisoptropy 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. phorescence is sensitive to motions in the range 10-6 < Tr < 100 seconds. Transient absorption anisotropy (5) and fluorescence recovery (6) are two additional methods that bridge the sensitivity ranges of the fluorescence and phosphorescence emission anisotropy methods. The use of EPR with nitroxide spin-labels to study the dynamics of lipids, DNA, proteins, and membrane bound proteins has been extensively reviewed (7, 8, 9, 10). Conventional linear EPR spectra of nitroxide spin-labels are sensitive to motions whose correlation times are in the range 10-"1 <,Tr < 10-6 seconds (11). The complementary saturation transfer EPR (ST-EPR

Research paper thumbnail of Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin

Biophysical Journal, 1993

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in ... more 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

Research paper thumbnail of Molecular distances from dipolar coupled spin-labels: the global analysis of multifrequency continuous wave electron paramagnetic resonance data

Biophysical Journal, 1997

For immobilized nitroxide spin-labels with a well-defined interprobe geometry, resolved dipolar s... more 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 N6-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 internitroxide 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.

Research paper thumbnail of The Sensitivity of Saturation Transfer Electron Paramagnetic Resonance Spectra to Restricted Amplitude Uniaxial Rotational Diffusion

Biophysical Journal, 2001

Computational methods have been developed to model the effects of constrained or restricted ampli... more 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 (VЈ 2) on the width (⌬) 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 s, the VЈ 2 signal is very sensitive to ⌬ in the range from 0 to 60°, marginally sensitive from 60°to 90°, and insensitive beyond 90°. Sensitivity to ⌬ depends on the correlation time for URD with higher sensitivity to large values of ⌬ 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.

Research paper thumbnail of The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy

Biophysical Journal, 1996

Research paper thumbnail of Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model

Biophysical Journal, 1995

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic r... more 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 Aand 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.

Research paper thumbnail of Structure of the cdb3-ankD34 Complex from Site Directed Spin Labeling Studies

Biophysical Journal, 2009

Algorithms have been developed for calculating the continuous wave electron paramagnetic resonanc... more Algorithms have been developed for calculating the continuous wave electron paramagnetic resonance (EPR) spectrum of a nitroxide spin label from a freeinduction decay (FID) derived from a molecular dynamics (MD) simulation. These algorithms have been successfully used to simulate the EPR spectra of two spin-labeled mutants of T4 lysozyme, T4L F153R1 and T4L K65R1 310a Monday,

Research paper thumbnail of Simulation of Nitroxide Electron Paramagnetic Resonance Spectra from Brownian Trajectories and Molecular Dynamics Simulations

Biophysical Journal, 2008

Research paper thumbnail of High Field/High Frequency Saturation Transfer Electron Paramagnetic Resonance Spectroscopy: Increased Sensitivity to Very Slow Rotational Motions

Biophysical Journal, 2004

Saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy has been employed to ch... more Saturation transfer electron paramagnetic resonance (ST-EPR) spectroscopy has been employed to characterize the very slow microsecond to millisecond rotational dynamics of a wide range of nitroxide spin-labeled proteins and other macromolecules in the past three decades. The vast majority of this previous work has been carried out on spectrometers that operate at X-band (;9 GHz) microwave frequency with a few investigations reported at Q-band (;34 GHz). EPR spectrometers that operate in the 94-250-GHz range and that are capable of making conventional linear EPR measurements on small aqueous samples have now been developed. This work addresses potential advantages of utilizing these same high frequencies for ST-EPR studies that seek to quantitatively analyze the very slow rotational dynamics of spin-labeled macromolecules. For example, the uniaxial rotational diffusion (URD) model has been shown to be particularly applicable to the study of the rotational dynamics of integral membrane proteins. Computational algorithms have been employed to define the sensitivity of ST-EPR signals at 94, 140, and 250 GHz to the correlation time for URD, to the amplitude of constrained URD, and to the orientation of the spin label relative to the URD axis. The calculations presented in this work demonstrate that these higher microwave frequencies provide substantial increases in sensitivity to the correlation time for URD, to small constraints in URD, and to the geometry of the spin label relative to the URD axis as compared with measurements made at X-band. Moreover, the calculations at these higher frequencies indicate sensitivity to rotational motions in the 1-100-ms time window, particularly at 250 GHz, thereby extending the slow motion limit for ST-EPR by two orders of magnitude relative to X-and Q-bands.

Research paper thumbnail of Structure of the Cytoplasmic Domain of Erythrocyte Band 3 Hereditary Spherocytosis Variant P327R:  Band 3 Tuscaloosa †

Biochemistry, 2007

Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band ... more Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electronelectron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.