Heinz-Jürgen Steinhoff

Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury

The nitrite anion is reduced to nitric oxide (NO•) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO• signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type (+/+) and knockout (−/−) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO• generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO• generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO• by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin+/+ but not in myoglobin−/− hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin+/+ mice, whereas in myoglobin−/− mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation. myoglobin knockout mice

Structural insights into the early steps of receptor—transducer signal transfer in archaeal phototaxis

Electron paramagnetic resonance‐based inter‐residue distance measurements between site‐directed spin‐labelled sites of sensory rhodopsin II (NpSRII) and its transducer NpHtrII from Natronobacterium pharaonis revealed a 2:2 complex with 2‐fold symmetry. The core of the complex is formed by the four transmembrane helices of a transducer dimer. Upon light excitation, the previously reported flap‐like movement of helix F of NpSRII induces a conformational change in the transmembrane domain of the transducer. The inter‐residue distance changes determined provide strong evidence for a rotary motion of the second transmembrane helix of the transducer. This helix rotation becomes uncoupled from changes in the receptor during the last step of the photocycle.

Spin labeling EPR.

Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin–transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.

The archaeal sensory rhodopsin II/transducer complex: a model for transmembrane signal transfer

Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two‐component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer complex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the receptor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mechanism might also be relevant for eubacterial chemoreceptor signalling.

Molecular orbital study of polarity and hydrogen bonding effects on the g and hyperfine tensors of site directed NO spin labelled bacteriorhodopsin

Semiempirical molecular orbital methods (PM3, INDO, ZINDO/S) have been used to calculate the effects of local electric fields and of hydrogen bonding on the g and hyperfine tensors of a nitroxide spin label model system. The results yield a linear correlation between the two principal tensor components g xx and A N zz at label sites of varying polarity. Hydrogen bonding with a single water molecule produces a constant shift of Δg xx ≅ −4 × 10−4. These theoretical results are used to interpret recent high field (3.4 T, 95 GHz) electron paramagnetic resonance investigations on site-directed spin labelled bacteriorhodopsin. This protein reveals a close correlation between proticity and polarity at the various label sites. The slope of the g xx versus A N zz dependence is affected strongly by polarity induced structural strains of the spin label.

Phosphorylation of a membrane curvature–sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif.

Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein- protein and protein-oligonucleotide interaction

Abstract Recent developments including pulse and multi-frequency techniques make the combination of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy an attractive approach for the study of protein-protein or protein-oligonucleotide interaction. Analysis of the spin label side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin label side chains allow the modeling of protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. Structural changes can be detected with millisecond time resolution. Inter- and intra-molecular distances are accessible in the range from approximately 0.5 to 8 nm by the combination of continuous wave and pulse EPR methods. Recent applications include the study of transmembrane substrate transport, membrane channel gating, gene regulation and signal transfer.

Structural Analysis of a HAMP Domain THE LINKER REGION OF THE PHOTOTRANSDUCER IN COMPLEX WITH SENSORY RHODOPSIN II

Sensory rhodopsin II, the photophobic receptor from Natronomonas pharaonis (NpSRII)5, forms a 2:2 complex with its cognate transducer (N. pharaonis halobacterial transducer of rhodopsins II (NpHtrII)) in lipid membranes. Light activation of NpSRII leads to a displacement of helix F, which in turn triggers a rotation/screw-like motion of TM2 in NpHtrII. This conformational change is thought to be transmitted through the membrane adjacent conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (HAMP domain) to the cytoplasmic signaling domain of the transducer. The architecture and function of the HAMP domain are still unknown. In order to obtain information on the structure and dynamics of this region, EPR experiments on a truncated transducer (NpHtrII157) and NpSRII, site-directed spin-labeled and reconstituted into purple membrane lipids, have been carried out. A nitroxide scanning involving residues in the transducer helix TM2, in the predicted AS-1 region, and at selected positions in the following connector and AS-2 regions of the HAMP domain has been performed. Accessibility and dynamics data allowed us to identify a helical region up to residue Ala94 in the AS-1 amphipathic sequence, followed by a highly dynamic domain protruding into the water phase. Additionally, transducer-transducer and transducer-receptor proximity relations revealed the overall architecture of the AS-1 sequences in the 2:2 complex, which are suggested to form a molten globular type of a coiled-coil bundle.

Transmembrane Signaling in the Maltose ABC Transporter MalFGK2-E PERIPLASMIC MalF-P2 LOOP COMMUNICATES SUBSTRATE AVAILABILITY TO THE ATP-BOUND MalK DIMER

ABC transporters are ubiquitous membrane proteins that translocate solutes across biological membranes at the expense of ATP. In prokaryotic ABC importers, the extracytoplasmic anchoring of the substrate-binding protein (receptor) is emerging as a key determinant for the structural rearrangements in the cytoplasmically exposed ATP-binding cassette domains and in the transmembrane gates during the nucleotide cycle. Here the molecular mechanism of such signaling events was addressed by electron paramagnetic resonance spectroscopy of spin-labeled ATP-binding cassette maltose transporter variants (MalFGK2-E). A series of doubly spin-labeled mutants in the MalF-P2 domain involving positions 92, 205, 239, 252, and 273 and one triple mutant labeled at positions 205/252 in P2 and 83 in the Q-loop of MalK were assayed. The EPR data revealed that the substrate-binding protein MalE is bound to the transporter throughout the transport cycle. Concomitantly with the three conformations of the ATP-binding cassette MalK2, three functionally relevant conformations are found also in the periplasmic MalF-P2 loop, strictly dependent on cytoplasmic nucleotide binding and periplasmic docking of liganded MalE to MalFG. The reciprocal communication across the membrane unveiled here gives first insights into the stimulatory effect of MalE on the ATPase activity, and it is suggested to be an important mechanistic feature of receptor-coupled ABC transporters.

Interresidual distance determination by four-pulse double electron-electron resonance in an integral membrane protein: the Na+/proline transporter PutP of Escherichia coli

Proximity relationships within three doubly spin-labeled variants of the Na+/proline transporter PutP of Escherichia coli were studied by means of four-pulse double electron-electron resonance spectroscopy. The large value of 4.8 nm for the interspin distance determined between positions 107 in loop 4 and 223 in loop 7 strongly supports the idea of these positions being located on opposite sides of the membrane. Significant smaller values of between 1.8 and 2.5 nm were found for the average interspin distances between spin labels attached to the cytoplasmic loops 2 and 4 (position 37 and 107) and loops 2 and 6 (position 37 and 187). The large distance distribution widths visible in the pair correlation functions reveal a high flexibility of the studied loop regions. An increase of the distance between positions 37 and 187 upon Na+ binding suggests ligand-induced structural alterations of PutP. The results demonstrate that four-pulse double electron-electron resonance spectroscopy is a powerful means to investigate the structure and conformational changes of integral membrane proteins reconstituted in proteoliposomes.