Lichi Shi

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). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

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. 0.2 ppm at fast spinning (28 kHz) in a protein back-exchanged at a level of 40%. Novel three-dimensional proton-detected chemical shift correlation spectroscopy allowed for the identification and resonance assignment of the solvent-exposed parts of the protein. Most of the observed residues are located at the membrane interface, but there are notable exceptions, particularly in helix G, where most of the residues are susceptible to H/D exchange. This helix contains Schiff base-forming Lys231, and many conserved polar residues in the extracellular half, such as Asn220, Tyr223, Asn224, Asp227, and Asn230. We proposed earlier that high mobility of the F-G loop may transiently expose a hydrophilic cavity in the extracellular half of the protein, similar to the one found in xanthorhodopsin. Solvent accessibility of helix G is in line with this hypothesis, implying that such a cavity may be a part of the proton-conducting pathway lined by this helix.

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. These experiments provided the assignments for 153 residues, with a particularly high density in the transmembrane regions ( approximately 74% of residues). The extent of assignments permitted a detailed examination of the secondary structure and dynamics in GPR. In particular, we present experimental evidence of mobility of the proteins termini and of the A-B, C-D, and F-G loops, the latter being possibly coupled to the GPR ion-transporting function.

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. We found an unusually strong coupling between the conformation of the retinal and the site of Glu132, the homolog of Asp96 of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp85 and Asp96 (Asp121 and Glu132). We assigned a pair of FTIR bands (positive at 1749 cm(-1) and negative at 1734 cm(-1)) to the protonation and deprotonation, respectively, of these carboxylic acids.

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. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully (13)C,(15)N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in beta-sheet content in actin, and increases in both alpha-helix and beta-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both alpha-helical and beta-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.

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. Most notably, the cytoplasmic half of helix G and the cytoplasmic ends of helices B and C exchange more extensively, probably as a result of their relative displacement in the activated state, allowing water to penetrate into the core of the protein. These light-induced rearrangements must provide the structural basis for the photosensory function of Anabaena sensory rhodopsin.

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. The sensitivity of the experiments and spectral resolution decrease with the increasing molecular weight, which makes setting SSNMR experiments in large proteins a much more challenging and demanding procedure. Here, we describe the protocol for the most common set of 3D magic angle spinning (MAS) SSNMR experiments. While the procedures described in the text are well known to SSNMR practitioners, we hope they will be of interest to scientists interested in extending their repertoire of biophysical techniques.

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. Amino acid sequence comparison of GR with other type I rhodopsin shows several conserved residues important for retinal binding and H+ pumping. In this study, the gene was expressed in Escherichia coli and bound all-trans retinal to form a pigment (λmax  = 544 nm at pH 7). The pKa of proton acceptor (Asp121) for the Schiff base, is approximately 5.9, so GR can translocate H+ under physiological conditions (pH 7.4). In order to prove the functional activity in the cell, pumping activity was measured in the sphaeroplast membranes of E. coli and one of Gloeobacter whole cell. The efficient proton pumping and rapid photocycle of GR strongly suggests that Gloeobacter rhodopsin functions as a proton pumping in its natural environment, probably compensating the shortage of energy generated by chlorophyll-based photosynthesis without thylakoids.

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 13C and 15N assignments of ASR reconstituted in lipids, obtained using two- and three-dimensional magic angle spinning solid state NMR spectroscopy on alternately 13C labeled samples. The obtained chemical shifts are used to characterize the protein backbone conformation. They suggest that lipid-reconstituted ASR has a fold generally similar to that seen in earlier X-ray studies, but with a number of important differences. SSNMR detects double conformations for a number of residues on the cytoplasmic side.

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 Cα carbons in the 3D NCACX experiment. In the proposed protocol, the refocusing of the Cα–CO homonuclear J-couplings is achieved by applying an off-resonance selective π pulse to the CO spectral region in the middle of Cα chemical shift evolution. As is commonly used in solution NMR, a compensatory echo period is used to refocus the unwanted chemical shift evolution of Cα spins, which takes place during the off-resonance selective pulse. The experiments were carried out on the β1 immunoglobulin binding domain of protein G (GB1). In GB1, such implementation results in significantly reduced line widths, and leads to an overall sensitivity enhancement.