Maryam Hashemi Shabestari

The complex of cytochrome f and plastocyanin from Nostoc sp. PCC 7119 is highly dynamic.

Cytochrome f (Cyt f) and plastocyanin (Pc) form a highly transient complex as part of the photosynthetic redox chain. The complex from Nostoc sp. PCC 7119 was studied by NMR relaxation spectroscopy with the aim of determining the orientation of Pc relative to Cyt f. Chemical‐shift‐perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc. The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.

Interaction of the amyloid β peptide with sodium dodecyl sulfate as a membrane-mimicking detergent.

The amyloid β (A β) peptide is important in the context of Alzheimer’s disease, since it is one of the major components of the fibrils that constitute amyloid plaques. Agents that can influence fibril formation are important, and of those, membrane mimics are particularly relevant, because the hydrophobic part of A β suggests a possible membrane activity of the peptide. We employed spin-label EPR to investigate the aggregation process of A β1–40 in the presence of the sodium dodecyl sulfate (SDS) detergent as a membrane-mimicking agent. In this work, the effect of SDS on A β is studied using two positions of spin label, the N-terminus and position 26. By comparing the two label positions, the effect of local mobility of the spin label is eliminated, revealing A β aggregation in the SDS concentration regime below the critical micelle concentration (CMC). We demonstrate that, at low SDS concentrations, the N-terminus of A β participates in the solubilization, most likely by being located at the particle–water interface. At higher SDS concentrations, an SDS-solubilized state that is a precursor to the one A β/micelle state above the CMC of SDS prevails. We propose that A β is membrane active and that aggregates include SDS. This study reveals the unique potential of EPR in studying A β aggregation in the presence of detergent.

Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29

The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.

In silico interpretation of cw-ESR at 9 and 95 GHz of mono- and bis- TOAC-labeled Aib-homopeptides in fluid and frozen acetonitrile.

In this paper, we address the interpretation of molecular properties of selected singly and doubly spin-labeled peptides from continuous-wave electron spin resonance (cw-ESR) spectroscopy. This study is performed by means of an integrated computational approach that merges a stochastic treatment of long-term dynamics to ad hoc methodologies for the calculation of structural properties. In particular, our method is based on (i) the determination of geometric and local magnetic parameters of the peptides by quantum mechanical density functional calculations by taking into account solvent contribution; (ii) the hydrodynamic evaluation of dissipative properties; and (iii) molecular dynamics including equilibrium distribution of molecular conformations. The system is then described by a stochastic Liouville equation in which the spin Hamiltonian for the two electron spins, interacting with each other and coupled to two (14)N nuclear spins, is coupled to the diffusive operator describing the time evolution of slow coordinates. cw-ESR spectra are simulated for selected peptides built from the non-natural α-aminoacids α-aminoisobutyric acid (Aib) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, we study the -Aib-TOAC-Aib- singly labeled tripeptide and the -Aib-TOAC-(Aib)(7)- singly labeled and -Aib-TOAC-(Aib)(5)-TOAC-Aib- doubly labeled nonapeptides. We show that good agreement is obtained with minimal resorting to fitting procedures, proving that the combination of sensitive ESR spectroscopy and sophisticated modeling is a powerful approach to the investigation of both molecular dynamics and 3D-structural properties.

Conformation and EPR characterization of rigid, 310-helical peptides with TOAC spin labels: Models for short distances

For 3D‐structure determination in biophysical systems EPR is rapidly gaining ground. Proteins labeled specifically with two nitroxide spin labels can be prepared, and several EPR methods are available for distance determination, which makes it possible to determine distance constraints. However, such methods require frozen solutions, potentially causing non‐physiological states of the sample. Here, we target spin– spin interaction in liquid solution at room temperature using rigid model compounds. A series of 310‐helical peptides, based on α‐aminoisobutyric acid (Aib), is synthesized with pairs of spin labels separated by three, four, and five amino acids. To avoid flexibility, the noncoded nitroxyl‐containing α‐amino acid TOAC that is rigidly connected with the peptide backbone, is used. The EPR spectra of the peptides show a decreasing amount of coupling between the two spin labels within this series. We suggest through‐bond interaction as the dominating mechanism for exchange interaction (J) and find a stronger J‐coupling than in the corresponding Ala‐based TOAC‐peptides investigated previously (Hanson, et al., J Am Chem Soc 1996, 118, 7618–7625). We speculate that stronger coupling in Aib‐ vs Ala‐ peptides is due to intrinsically stronger through‐bond interaction in the Aib‐based peptides.

In situ, Reversible Gating of a Mechanosensitive Ion Channel through Protein-Lipid Interactions

Understanding the functioning of ion channels, as well as utilizing their properties for biochemical applications requires control over channel activity. Herein we report a reversible control over the functioning of a mechanosensitive ion channel by interfering with its interaction with the lipid bilayer. The mechanosensitive channel of large conductance from Escherichia coli is reconstituted into liposomes and activated to its different sub-open states by titrating lysophosphatidylcholine (LPC) into the lipid bilayer. Activated channels are closed back by the removal of LPC out of the membrane by bovine serum albumin (BSA). Electron paramagnetic resonance spectra showed the LPC-dose-dependent gradual opening of the channel pore in the form of incrementally increasing spin label mobility and decreasing spin-spin interaction. A method to reversibly open and close mechanosensitive channels to distinct sub-open conformations during their journey from the closed to the fully open state enables detailed structural studies to follow the conformational changes during channel functioning. The ability of BSA to revert the action of LPC opens new perspectives for the functional studies of other membrane proteins that are known to be activated by LPC.

Integrated Computational Approach to the Electron Paramagnetic Resonance Characterization of Rigid 310-Helical Peptides with TOAC Nitroxide Spin Labels

We address the interpretation, via an integrated computational approach, of the experimental continuous-wave electron paramagnetic resonance (cw-EPR) spectra of a complete set of conformationally highly restricted, stable 310-helical peptides from hexa- to nonamers, each bis-labeled with nitroxide radical-containing TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) residues. The usefulness of TOAC for this type of analysis has been shown already to be due to its cyclic piperidine side chain, which is rigidly connected to the peptide backbone α-carbon. The TOAC α-amino acids are separated by two, three, four, and five intervening residues. This set of compounds has allowed us to modulate both the radical···radical distance and the relative orientation parameters. To further validate our conclusion, a comparative analysis has been carried out on three singly TOAC-labeled peptides of similar main-chain length.