As many key proteins evade crystallization and remain too large for nuclear magnetic resonance spectroscopy, electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling offers an alternative approach for obtaining structural information. Such information must be translated into geometric restraints to be used in computer simulations. Here, distances between spin labels are converted into distance ranges between beta carbons by using a "motion-on-a-cone" model, and a linear-correlation model links spin-label accessibility to the number of neighboring residues. This approach was tested on T4-lysozyme and alphaA-crystallin with the de novo structure prediction algorithm Rosetta. The results demonstrate the feasibility of obtaining highly accurate, atomic-detail models from EPR data by yielding 1.0 A and 2.6 A full-atom models, respectively. Distance restraints between amino acids far apart in sequence but close in space are most valuable for structure determination. The approach can be extended to other experimental techniques such as fluorescence spectroscopy, substituted cysteine accessibility method, or mutational studies.
Driven by the energy of ATP binding and hydrolysis, ATP binding cassette (ABC) transporters alternate between inward-and outward-facing conformations allowing vectorial movement of substrates. Conflicting models have been proposed to describe the conformational motion underlying this switch in access of the transport pathway. One model, based on three crystal structures of the lipid flippase MsbA, envisions a large amplitude motion that disengages the nucleotide binding domains and repacks the transmembrane helices. To test this model and place the crystal structures in a mechanistic context, we use spin labeling and Double Electron Electron Resonance (DEER) spectroscopy to define the nature and amplitude of MsbA conformational change during ATP hydrolysis cycle. For this purpose, spin labels were introduced at sites selected to provide a distinctive pattern of distance changes unique to the crystallographic transformation. Distance changes in liposomes, induced by the transition from nucleotide-free MsbA to the highest energy intermediate, fit a simple pattern whereby residues on the cytoplasmic side undergo 20-30Å closing motion while a 7-10Å opening motion is observed on the extracellular side. The transmembrane helices undergo relative movement to create the outward opening consistent with that implied by the crystal structures. DEER distance distributions reveal asymmetric backbone flexibility on the two sides of the transporter that correlates with asymmetric opening of the substrate binding chamber. Together with extensive accessibility analysis, our results suggest that these structures capture features of the motion that couples ATP energy expenditure to work providing a framework for the mechanism of substrate transport. KeywordsABC transporter; MsbA; double electron-electron resonance (DEER); site-directed spin labeling; electron paramagnetic resonance Active transporters transduce various forms of energy into the mechanical work of substrate translocation. For the class of ATP binding cassettes (ABC) transporters, ATP energy drives protein conformational motions to carry molecules ranging from small ions to large polypeptides across membranes 1,2 . The spectrum of these movements, their amplitudes and *Corresponding author. Mailing address for Hassane S. Mchaourab: 741 Light Hall, 2215 Garland Ave., Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA, hassane.mchaourab@vanderbilt.edu. ⊥ Current address: Dept. Scienze Chimiche, Universita' degli studi di Padova, Padova, Italy.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers tha...
We measured the amplitude of conformational motion in the ATP-binding cassette (ABC) transporter MsbA upon lipopolysaccharide (LPS) binding and following ATP turnover by pulse double electron-electron resonance and fluorescence homotransfer. The distance constraints from both methods reveal large-scale movement of opposite signs in the periplasmic and cytoplasmic part of the transporter upon ATP hydrolysis. LPS induces distinct structural changes that are inhibited by trapping of the transporter in an ATP post-hydrolysis intermediate. The formation of this intermediate involves a 33-Å distance change between the two ABCs, which is consistent with a dimerization-dissociation cycle during transport that leads to their substantial separation in the absence of nucleotides. Our results suggest that ATP-powered transport entails LPS sequestering into the open cytoplasmic chamber prior to its translocation by alternating access of the chamber, made possible by 10–20-Å conformational changes.
We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2′,7′-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q2) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q1 as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decylubiquinone. This behaviour relates the prooxidant CoQ1 activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH–DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron–sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.
Human small heat shock protein 27 (Hsp27) undergoes concentration-dependent equilibrium dissociation from an ensemble of large oligomers to a dimer. This phenomenon plays a critical role in Hsp27 chaperone activity in vitro enabling high affinity binding to destabilized proteins. In vivo dissociation, which is regulated by phosphorylation, controls Hsp27 role in signaling pathways. In this study, we explore the sequence determinants of Hsp27 dissociation and define the structural basis underlying the increased affinity of Hsp27 dimers to client proteins. A systematic cysteine mutagenesis is carried out to identify residues in the N-terminal domain important for the equilibrium between Hsp27 oligomers and dimers. In addition, spin labels were attached to the cysteine mutants to enable electron paramagnetic resonance (EPR) analysis of residue environment and solvent accessibility in the context of the large oligomers, upon dissociation to the dimer, and following complex formation with the model substrate T4 Lysozyme (T4L). The mutagenic analysis identifies residues that modulate the equilibrium dissociation in favor of the dimer. EPR analysis reveals that oligomer dissociation disrupts subunit contacts leading to the exposure of Hsp27 N-terminal domain to the aqueous solvent. Moreover, regions of this domain are highly dynamic with no evidence of a packed core. Interaction between T4L and sequences in this domain is inferred from transition of spin labels to a buried environment in the substrate/Hsp27 complex. Together, the data provides the first structural analysis of sHSP dissociation and supports a model of chaperone activity wherein unstructured and highly flexible regions in the N-terminal domain are critical for substrate binding.
The region 35-43 of human alpha-Synuclein bound to small unilamellar lipid vesicles and to sodium dodecyl sulfate micelles has been investigated by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The distance distributions obtained from spectral fitting have been analyzed on the basis of the allowed rotamers of the spin-label side-chain. Very similar results have been obtained in the two environments: an unbroken helical structure of the investigated region can be ruled out. The distance distributions are rather compatible with the presence of conformational disorder, in agreement with previous findings for micelle-bound alpha-Synuclein. The propensity for helix breaking is confirmed by molecular dynamics simulations.
Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35°C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer.
Bicelles are model membrane systems that can be macroscopically oriented in a magnetic field at physiological temperature. The macroscopic orientation of bicelles allows to detect, by means of magnetic resonance spectroscopies, small changes in the order of the bilayer caused by solutes interacting with the membrane. These changes would be hardly detectable in isotropic systems such as vesicles or micelles. The aim of this work is to show that bicelles represent a convenient tool to investigate the behavior of antimicrobial peptides (AMPs) interacting with membranes, using electron paramagnetic resonance (EPR) spectroscopy. We performed the EPR experiments on spin-labeled bicelles using various AMPs of different length, charge, and amphipathicity: alamethicin, trichogin GA IV, magainin 2, HP(2-20), and HPA3. We evaluated the changes in the order parameter of the spin-labeled lipids as a function of the peptide-to-lipid ratio. We show that bicelles labeled at position 5 of the lipid chains are very sensitive to the perturbation induced by the AMPs even at low peptide concentrations. Our study indicates that peptides that are known to disrupt the membrane by different mechanisms (i.e., alamethicin vs magainin 2) show very distinct trends of the order parameter as a function of peptide concentration. Therefore, spin-labeled bicelles proved to be a good system to evaluate the membrane disruption mechanism of new AMPs.
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