Cyanobacterial light-driven proton pump, gloeobacter rhodopsin: complementarity between rhodopsin-based energy production and photosynthesis.

A homologue of type I rhodopsin was found in the unicellular Gloeobacter violaceus PCC7421, which is believed to be primitive because of the lack of thylakoids and peculiar morphology of phycobilisomes. The Gloeobacter rhodopsin (GR) gene encodes a polypeptide of 298 amino acids. This gene is localized alone in the genome unlike cyanobacterium Anabaena opsin, which is clustered together with 14 kDa transducer gene.

Tracing an allosteric pathway regulating the activity of the HslV protease.

The HslU-HslV complex functions as a bacterial proteasome, degrading substrate polypeptides to preserve cellular homeostasis. Here, we use methyl-Transverse Relaxation-Optimized Spectroscopy (TROSY) and highly deuterated, methyl-protonated samples to study the 230 kDa dodecameric HslV protease component that is structurally homologous to the stacked pair of β7-rings of the proteasome. Chemical shift assignments for over 95% of the methyl groups are reported.

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR).

Solid-state NMR ¹³C and ¹⁵N resonance assignments of a seven-transmembrane helical protein Anabaena Sensory Rhodopsin.

Anabaena Sensory Rhodopsin (ASR) is a unique microbial rhodopsin that displays photocromism, interacts with soluble transducer, and may be involved in gene regulation. Here we report nearly complete spectroscopic (13)C and (15)N assignments of ASR reconstituted in lipids, obtained using two- and three-dimensional magic angle spinning solid state NMR spectroscopy on alternately (13)C labeled samples. The obtained chemical shifts are used to characterize the protein backbone conformation.

Magic angle spinning solid-state NMR experiments for structural characterization of proteins.

Solid-state nuclear magnetic resonance (SSNMR) has become a prominent method in biology and is suitable for the characterization of insoluble proteins and protein aggregates such as amyloid fibrils, membrane-lipid complexes, and precipitated proteins. Often, the initial and the most critical step is to obtain spectroscopic assignments, that is, to determine chemical shifts of individual atoms. The procedures for SSNMR spectroscopic assignments are now well established for small microcrystalline proteins, where high signal-to-noise can be obtained.

Proton-detected solid-state NMR reveals intramembrane polar networks in a seven-helical transmembrane protein proteorhodopsin.

We used high-resolution proton-detected multidimensional NMR to study the solvent-exposed parts of a seven-helical integral membrane proton pump, proteorhodopsin (PR). PR samples were prepared by growing the apoprotein on fully deuterated medium and reintroducing protons to solvent-accessible sites through exchange with protonated buffer. This preparation leads to NMR spectra with proton resolution down to ca.

Site-specific solid-state NMR detection of hydrogen-deuterium exchange reveals conformational changes in a 7-helical transmembrane protein.

Solid-state NMR spectroscopy is an efficient tool for following conformational dynamics of membrane proteins at atomic resolution. We used this technique for the site-specific detection of light-induced hydrogen-deuterium exchange in the lipid-embedded heptahelical transmembrane photosensor Anabaena sensory rhodopsin to pinpoint the location of its conformational changes upon activation. We show that the light-induced conformational changes result in a dramatic, but localized, increase in the exchange in the transmembrane regions.

Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive.

Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C,15N-labeled peptides and proteins.

In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly (13)C,(15)N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory.

Solid-state NMR study of proteorhodopsin in the lipid environment: secondary structure and dynamics.

Proteorhodopsins are typical retinal-binding light-driven proton pumps of heptahelical architecture widely distributed in marine and freshwater bacteria. Recently, we have shown that green proteorhodopsin (GPR) can be prepared in a lipid-bound state that gives well-resolved magic angle spinning (MAS) NMR spectra in samples with different patterns of reverse labelling. Here, we present 3D and 4D sequential chemical shift assignments identified through experiments conducted on a uniformly (13)C,(15)N-labelled sample.

Three-dimensional solid-state NMR study of a seven-helical integral membrane proton pump--structural insights.

Proteorhodopsin (PR) is a recently discovered ubiquitous eubacterial retinal-binding light-driven proton pump. Almost 1000 PR variants are widely distributed in species of marine and freshwater bacteria, suggesting PR's important photobiological role. PR is a typical seven-transmembrane alpha-helical membrane protein and as such poses a significant challenge to structural studies.

The photocycle and proton translocation pathway in a cyanobacterial ion-pumping rhodopsin.

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation.

Induced secondary structure and polymorphism in an intrinsically disordered structural linker of the CNS: solid-state NMR and FTIR spectroscopy of myelin basic protein bound to actin.

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia.

Resolution enhancement by homonuclear J-decoupling: application to three-dimensional solid-state magic angle spinning NMR spectroscopy.

We describe a simple protocol to achieve homonuclear J-decoupling in the indirect dimensions of multidimensional experiments, and to enhance spectral resolution of the backbone Calpha carbons in the 3D NCACX experiment. In the proposed protocol, the refocusing of the Calpha-CO homonuclear J-couplings is achieved by applying an off-resonance selective pi pulse to the CO spectral region in the middle of Calpha chemical shift evolution. As is commonly used in solution NMR, a compensatory echo period is used to refocus the unwanted chemical shift evolution of Calpha spins, which takes place during the off-resonance selective pulse.

Structural basis of diversification of fungal retinal proteins probed by site-directed mutagenesis of Leptosphaeria rhodopsin.

Numerous fungal genomes encode homologs of bacteriorhodopsin (BR), but only two fungal rhodopsins were overexpressed and characterized spectroscopically. Neurospora rhodopsin (NR) is a slow-cycling sensory rhodopsin-like protein, while Leptosphaeria rhodopsin (LR) is a BR-like proton pump. Recently, we found that a conservative replacement of the cytoplasmic proton donor Asp150 by Glu converts LR into an NR-like protein.

Conformational coupling between the cytoplasmic carboxylic acid and the retinal in a fungal light-driven proton pump.

Many fungal rhodopsins, eukaryotic structural homologues of the archaeal light-driven proton pump bacteriorhodopsin, have been discovered in the course of genome sequencing projects. Recently, two fungal rhodopsins were characterized in vitro and exhibited very different photochemical behavior. Neurospora rhodopsin possesses a slow photocycle and shows no ion transport, reminiscent of sensory rhodopsins, while Leptosphaeria rhodopsin has a fast bacteriorhodopsin-like photocycle and pumps protons light-dependently.

Cytoplasmic shuttling of protons in anabaena sensory rhodopsin: implications for signaling mechanism.

It was found recently that Anabaena sensory rhodopsin (ASR), which possibly serves as a photoreceptor for chromatic adaptation, interacts with a soluble cytoplasmic transducer. The X-ray structure of the transducer-free protein revealed an extensive hydrogen-bonded network of amino acid residues and water molecules in the cytoplasmic half of ASR, in high contrast to its haloarchaeal counterparts. Using time-resolved spectroscopy of the wild-type and mutant ASR in the visible and infrared ranges, we tried to determine whether this hydrogen-bonded network is used to translocate protons and whether those proton transfers are important for interaction with the transducer.

Leptosphaeria rhodopsin: bacteriorhodopsin-like proton pump from a eukaryote.

Bacteriorhodopsin-like proteins provide archaea and eubacteria with a unique bioenergetic pathway comprising light-driven transmembrane proton translocation by a single retinal-binding protein. Recently, homologous proteins were found to perform photosensory functions in lower eukaryotes, but no active ion transport by eukaryotic rhodopsins was detected. By demonstrating light-driven proton pumping in a fungal rhodopsin from Leptosphaeria maculans, we present a case of a retinal-based proton transporter from a eukaryote.