Eric J. Hustedt

Vanderbilt University, Нашвилл, Michigan, United States

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Publications (47)216.76 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3 Å from a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3 Å distance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.
    Journal of Magnetic Resonance 10/2014; DOI:10.1016/j.jmr.2014.09.020 · 2.32 Impact Factor
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    ABSTRACT: We report here specialized functions incorporated recently in the rigid-body docking software toolkit TagDock to utilize electron paramagnetic resonance derived (EPR-derived) interresidue distance measurements and spin-label accessibility data. The TagDock package extensions include a custom methanethiosulfonate spin label rotamer library to enable explicit, all-atom spin-label side-chain modeling and scripts to evaluate spin-label surface accessibility. These software enhancements enable us to better utilize the biophysical data routinely available from various spin-labeling experiments. To illustrate the power and utility of these tools, we report the refinement of an ankyrin:CDB3 complex model that exhibits much improved agreement with the EPR distance measurements, compared to model structures published previously.
    The Journal of Physical Chemistry B 04/2014; 118(18). DOI:10.1021/jp4099705 · 3.30 Impact Factor
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    ABSTRACT: The multi-domain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using site-directed spin labeling method of the electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX 'BH3-in-groove homodimer.' Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK 'BH3-in-groove homodimers' in the oligomeric BAK. This novel interface involves the C-termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a 'worm hole.'
    Journal of Biological Chemistry 12/2013; 289(5). DOI:10.1074/jbc.M113.526806 · 4.57 Impact Factor
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    ABSTRACT: The 99-residue transmembrane C-terminal domain (C99, also known as β-CTF) of the amyloid precursor protein (APP) is the product of the β-secretase cleavage of the full-length APP and is the substrate for γ-secretase cleavage. The latter cleavage releases the amyloid-β polypeptides that are closely associated with Alzheimer's disease. C99 is thought to form homodimers; however, the free energy in favor of dimerization has not previously been quantitated. It was also recently documented that cholesterol forms a 1:1 complex with monomeric C99 in bicelles. Here, the affinities for both homodimerization and cholesterol binding to C99 were measured in bilayered lipid vesicles using both electron paramagnetic resonance (EPR) and Förster resonance energy transfer (FRET) methods. Homodimerization and cholesterol binding were seen to be competitive processes that center on the transmembrane G700XXXG704XXXG708 glycine-zipper motif and adjacent Gly709. On one hand, the observed Kd for cholesterol binding (Kd = 2.7 ± 0.3 mol %) is on the low end of the physiological cholesterol concentration range in mammalian cell membranes. On the other hand, the observed Kd for homodimerization (Kd = 0.47 ± 0.15 mol %) likely exceeds the physiological concentration range for C99. These results suggest that the 1:1 cholesterol/C99 complex will be more highly populated than C99 homodimers under most physiological conditions. These observations are of relevance for understanding the γ-secretase cleavage of C99.
    Biochemistry 07/2013; 52(30). DOI:10.1021/bi400735x · 3.01 Impact Factor
  • Alzheimer's and Dementia 07/2013; 9(4):P365. DOI:10.1016/j.jalz.2013.05.705 · 17.47 Impact Factor
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    ABSTRACT: The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion across the plasma membrane of cardiomyocytes after the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to its intracellular domain, which contains two structurally homologous Ca2+ binding domains denoted as CBD1 and CBD2. NMR and x-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have shown how Ca2+ binding affects their structures and motional dynamics. However, structural information on the entire Ca2+ binding domain, denoted CBD12, and how binding of Ca2+ alters its structure and dynamics is more limited. Site-directed spin labeling has been employed in this work to address these questions. Electron paramagnetic resonance measurements on singly labeled constructs of CBD12 have identified the regions that undergo changes in dynamics as a result of Ca2+ binding. Double electron-electron resonance (DEER) measurements on doubly labeled constructs of CBD12 have shown that the β-sandwich regions of the CBD1 and CBD2 domains are largely insensitive to Ca2+ binding and that these two domains are widely separated at their N and C termini. Interdomain distances measured by DEER have been employed to construct structural models for CBD12 in the presence and absence of Ca2+. These models show that there is not a major change in the relative orientation of the two Ca2+ binding domains as a result of Ca2+ binding in the NCX1.1 isoform. Additional measurements have shown that there are significant changes in the dynamics of the F-G loop region of CBD2 that merit further characterization with regard to their possible involvement in regulation of NCX1.1 activity.
    Journal of Biological Chemistry 12/2012; 288(6). DOI:10.1074/jbc.M112.423293 · 4.57 Impact Factor
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    ABSTRACT: C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.
    Science 06/2012; 336(6085):1168-71. DOI:10.1126/science.1219988 · 31.48 Impact Factor
  • Aaron W Kittell · Eric J Hustedt · James S Hyde
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    ABSTRACT: Site-directed spin-labeling electron paramagnetic resonance (SDSL EPR) provides insight into the local structure and motion of a spin probe strategically attached to a molecule. When a second spin is introduced to the system, macromolecular information can be obtained through measurement of inter-spin distances either by continuous wave (CW) or pulsed electron double resonance (ELDOR) techniques. If both methodologies are considered, inter-spin distances of 8-80 Å can be experimentally determined. However, there exists a region at the upper limit of the conventional X-band (9.5 GHz) CW technique and the lower limit of the four-pulse double electron-electron resonance (DEER) experiment where neither method is particularly reliable. The work presented here utilizes L-band (1.9 GHz) in combination with non-adiabatic rapid sweep (NARS) EPR to address this opportunity by increasing the upper limit of the CW technique. Because L-band linewidths are three to seven times narrower than those at X-band, dipolar broadenings that are small relative to the X-band inhomogeneous linewidth become observable, but the signal loss, due to the frequency dependence of the Boltzmann factor, has made L-band especially challenging. NARS has been shown to increase sensitivity by a factor of five, and overcomes much of this loss, making L-band distance determination more feasible. Two different systems are presented, and distances of 18-30 Å have been experimentally determined at physiologically relevant temperatures. Measurements are in excellent agreement with a helical model and values determined by DEER.
    Journal of Magnetic Resonance 05/2012; 221:51-6. DOI:10.1016/j.jmr.2012.05.006 · 2.32 Impact Factor
  • Suzanne Brandon · Albert H Beth · Eric J Hustedt
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    ABSTRACT: Double Electron-Electron Resonance (DEER) has emerged as a powerful technique for measuring long range distances and distance distributions between paramagnetic centers in biomolecules. This information can then be used to characterize functionally relevant structural and dynamic properties of biological molecules and their macromolecular assemblies. Approaches have been developed for analyzing experimental data from standard four-pulse DEER experiments to extract distance distributions. However, these methods typically use an a priori baseline correction to account for background signals. In the current work an approach is described for direct fitting of the DEER signal using a model for the distance distribution which permits a rigorous error analysis of the fitting parameters. Moreover, this approach does not require a priori background correction of the experimental data and can take into account excluded volume effects on the background signal when necessary. The global analysis of multiple DEER data sets is also demonstrated. Global analysis has the potential to provide new capabilities for extracting distance distributions and additional structural parameters in a wide range of studies.
    Journal of Magnetic Resonance 05/2012; 218:93-104. DOI:10.1016/j.jmr.2012.03.006 · 2.32 Impact Factor
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    ABSTRACT: A membrane alignment technique has been used to measure the distance between two TOAC nitroxide spin labels on the membrane-spanning M2δ, peptide of the nicotinic acetylcholine receptor (AChR), via CW-EPR spectroscopy. The TOAC-labeled M2δ peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 14.6 Å from a dual TOAC-labeled AChR M2δ peptide at positions 7 and 13 that closely matches with the 14.5 Å distance obtained from a model of the labeled AChR M2δ peptide. In addition, the angles determining the relative orientation of the two nitroxides have been determined, and the results compare favorably with molecular modeling. The global analysis of the data from the aligned samples gives much more precise estimates of the parameters defining the geometry of the two labels than can be obtained from a randomly dispersed sample.
    The Journal of Physical Chemistry B 03/2012; 116(12):3866-73. DOI:10.1021/jp212272d · 3.30 Impact Factor
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    ABSTRACT: The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.
    Journal of Biological Chemistry 06/2011; 286(23):20746-57. DOI:10.1074/jbc.M111.230326 · 4.57 Impact Factor
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    ABSTRACT: The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13–24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18–20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7–12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.
    Journal of Biological Chemistry 06/2011; 286(23):20746-20757. · 4.57 Impact Factor
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    Biophysical Journal 02/2011; 100(3). DOI:10.1016/j.bpj.2010.12.1001 · 3.97 Impact Factor
  • Biophysical Journal 02/2011; 100(3). DOI:10.1016/j.bpj.2010.12.996 · 3.97 Impact Factor
  • Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.2155 · 3.97 Impact Factor
  • Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.2154 · 3.97 Impact Factor
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    ABSTRACT: Conformational flexibility in nucleic acids provides a basis for complex structures, binding, and signaling. One-base bulges directly neighboring single-base mismatches in nucleic acids can be present in a minimum of two distinct conformations, complicating the examination of the thermodynamics by calorimetry or UV-monitored melting techniques. To provide additional information about such structures, we demonstrate how electron paramagnetic resonance (EPR) active spin-labeled base analogues, base-specifically incorporated into the DNA, are monitors of the superposition of different bulge-mismatch conformations. EPR spectra provide information about the dynamic environments of the probe. This information is cast in terms of "dynamic signatures" that have an underlying basis in structural variations. By examining the changes in the equilibrium of the different states across a range of temperatures, the enthalpy and entropy of the interconversion among possible conformations can be determined. The DNA constructs with a single bulge neighboring a single-base mismatch ("bulge-mismatches") may be approximately modeled as an equilibrium between two possible conformations. This structural information provides insight into the local composition of the bulge-mismatch sequences. Experiments on the bulge-mismatches show that basepairing across the helix can be understood in terms of purine and pyrimidine interactions, rather than specific bases. Measurements of the enthalpy and entropy of formation for the bulge-mismatches by differential scanning calorimetry and UV-monitored melting confirm that the formation of bulge-mismatches is in fact more complicated than a simple two-state process, consistent with the base-specific spectral data that bulge-mismatches exist in multiple conformations in the premelting temperature region. We find that the calculations with the nearest-neighbor (NN) model for the two likely conformations do not correlate well with the populations of structures and thermodynamic parameters inferred from the base-specific EPR dynamics probe. We report that the base-specific spin probes are able to identify a bistable, temperature dependent, switching between conformations for a particular complex bulged construct.
    The Journal of Physical Chemistry B 04/2009; 113(9):2664-75. DOI:10.1021/jp808260b · 3.30 Impact Factor
  • Biophysical Journal 02/2009; 96(3). DOI:10.1016/j.bpj.2008.12.3808 · 3.97 Impact Factor
  • Kelli N. Kazmier · Terry P. Lybrand · Eric J. Hustedt
    Biophysical Journal 02/2009; 96(3). DOI:10.1016/j.bpj.2008.12.1549 · 3.97 Impact Factor
  • Biophysical Journal 02/2009; 96(3). DOI:10.1016/j.bpj.2008.12.1544 · 3.97 Impact Factor

Publication Stats

786 Citations
216.76 Total Impact Points

Institutions

  • 1993–2014
    • Vanderbilt University
      • • Department of Biochemistry
      • • Department of Molecular Physiology and Biophysics
      Нашвилл, Michigan, United States
  • 1990–2004
    • University of Washington Seattle
      • Department of Chemistry
      Seattle, WA, United States
  • 1997
    • University of Illinois, Urbana-Champaign
      Urbana, Illinois, United States
  • 1990–1991
    • University of New Brunswick
      • Department of Physics
      Fredericton, New Brunswick, Canada