Resonance Raman measurement of macular carotenoids in the living human eye

There is growing evidence that high levels of the macular xanthophyll carotenoids lutein and zeaxanthin may be protective against visual loss from age-related macular degeneration. To study this protective effect further, it is important to measure macular carotenoid levels noninvasively in a wide variety of subjects. We have developed and validated resonance Raman spectroscopy as a sensitive and specific objective method to measure macular carotenoid levels in the living human eye. In this minireview, the principles and implementation of ocular carotenoid resonance Raman spectroscopy are reviewed, and the results of observational cross-sectional studies and of prospective supplementation studies on subjects with and without macular pathology are summarized. We have recently extended this technology to an imaging mode which will further enhance our understanding of the roles of lutein and zeaxanthin in normal macular function and in the prevention of age-related visual loss.     

Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage."     

Subunit Organization in the TatA Complex of the Twin Arginine Protein Translocase: A SITE-DIRECTED EPR SPIN LABELING STUDY*

The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile¹² and Val¹⁴ are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile¹² on one subunit and Val¹⁴ on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.     

The Lipid-binding Domain of Wild Type and Mutant α-Synuclein: COMPACTNESS AND INTERCONVERSION BETWEEN THE BROKEN AND EXTENDED HELIX FORMS*

α-Synuclein (αS) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits, and by the existence of three αS point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function of αS is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles, αS undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated helices. Membrane-bound conformations of αS likely mediate the function of the protein, but may also play a role in the aggregation and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the detergent- and membrane-bound conformations of αS, as well as factors that govern the transition of the protein between the extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all three PD-linked αS mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition, we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether the protein adopts the broken or extended helix conformation.     

31P saturation transfer spectroscopy predicts differential intracellular macromolecular association of ATP and ADP in skeletal muscle

The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo ³¹P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in ³¹P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of ³¹P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of β-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP ↔ ATP exchange, in which ADP is only transiently present in the cytosol.     

Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl? Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl⁻ and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl⁻ secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca²⁺ from the endoplasmic reticulum (ER), lowering [Ca²⁺] in the ER and thereby activating the Ca²⁺-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca²⁺] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl⁻ current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca²⁺ buffer that lowers [Ca²⁺] in the ER similar to the effect of 3O-C12 also increased cAMP and I_Cl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl⁻ and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca²⁺] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl⁻ and fluid secretion.     

Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Ras family small GTPases assume two interconverting conformations, “inactive” state 1 and “active” state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5′-(β,γ-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the ³¹P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.     

The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase seperation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane.     

Structure,Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.     

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices α5 and α6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.