A Detailed View on the (Re)isomerization Dynamics in Microbial Rhodopsins Using Complementary Near-UV and IR Readouts.

Isomerization is a key process in many (bio)chemical systems. In microbial rhodopsins, the photoinduced isomerization of the all-trans retinal to the 13-cis isomer initiates a cascade of structural changes of the protein. The interplay between these changes and the thermal relaxation of the isomerized retinal is one of the crucial determinants for rhodopsin functionality.

Structural response of G protein binding to the cyclodepsipeptide inhibitor FR900359 probed by NMR spectroscopy.

The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of G proteins by blocking GDP/GTP exchange. The inhibitor binding site of G has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR.

Molecular mechanisms and evolutionary robustness of a color switch in proteorhodopsins.

Proteorhodopsins are widely distributed photoreceptors from marine bacteria. Their discovery revealed a high degree of evolutionary adaptation to ambient light, resulting in blue- and green-absorbing variants that correlate with a conserved glutamine/leucine at position 105. On the basis of an integrated approach combining sensitivity-enhanced solid-state nuclear magnetic resonance (ssNMR) spectroscopy and linear-scaling quantum mechanics/molecular mechanics (QM/MM) methods, this single residue is shown to be responsible for a variety of synergistically coupled structural and electrostatic changes along the retinal polyene chain, ionone ring, and within the binding pocket.

Probing the allosteric NBD-TMD crosstalk in the ABC transporter MsbA by solid-state NMR.

The ABC transporter MsbA plays a critical role in Gram-negative bacteria in the regulation of the outer membrane by translocating core-LPS across the inner membrane. Additionally, a broad substrate specificity for lipophilic drugs has been shown. The allosteric interplay between substrate binding in the transmembrane domains and ATP binding and turnover in the nucleotide-binding domains must be mediated via the NBD/TMD interface.

Automated Fragmentation Quantum Mechanical Calculation of N and C Chemical Shifts in a Membrane Protein.

In this work, we developed an accurate and cost-effective automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) method to calculate the chemical shifts of N and C of membrane proteins. The convergence of the AF-QM/MM method was tested using rhodopsin 2 as a test case. When the distance threshold of the QM region is equal to or larger than 4.

Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles.

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2.

Rhodamine-Sensitized Two-Photon Activation of a Red Light-Absorbing BODIPY Photocage.

Two-photon (2P) activatable probes are of high value in biological and medical chemistry since near infrared (NIR) light can penetrate deeply even in blood-perfused tissue and due to the intrinsic three-dimensional activation properties. Designing two-photon chromophores is challenging. However, the two-photon absorption qualities of a photocage can be improved with an intramolecular sensitizer, which transfers the absorbed light onto the cage.

Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2.

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion-pumping characteristics by mutations is therefore of great interest.

Solid-State NMR Spectroscopy on Microbial Rhodopsins.

Microbial rhodopsins represent the most abundant phototrophic systems known today. A similar molecular architecture with seven transmembrane helices and a retinal cofactor linked to a lysine in helix 7 enables a wide range of functions including ion pumping, light-controlled ion channel gating, or sensing. Deciphering their molecular mechanisms therefore requires a combined consideration of structural, functional, and spectroscopic data in order to identify key factors determining their function.

Applications of Cell-Free Synthesized Membrane Protein Precipitates.

Cell-free protein expression systems are new core platforms for membrane protein synthesis. Expression in the presence of supplied artificial hydrophobic environments such as nanomembranes or micelles allows the co-translational solubilization and folding of membrane proteins. In the absence of hydrophobic compounds, the synthesized membrane proteins quantitatively precipitate, while frequently still retaining a significant part of folded structural elements.

Room-temperature dynamic nuclear polarization enhanced NMR spectroscopy of small biological molecules in water.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful and popular technique for probing the molecular structures, dynamics and chemical properties. However the conventional NMR spectroscopy is bottlenecked by its low sensitivity. Dynamic nuclear polarization (DNP) boosts NMR sensitivity by orders of magnitude and resolves this limitation.

MsbA: an ABC transporter paradigm.

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range.

Transient Near-UV Absorption of the Light-Driven Sodium Pump Rhodopsin 2: A Spectroscopic Marker for Retinal Configuration.

We report a transient signature in the near-UV absorption of rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all- to 13- isomerization of retinal and decays with the reisomerization to all- in the late photocycle, making it a promising marker band for retinal configuration. Hybrid quantum mechanics/molecular mechanics simulations show that the near-UV absorption signal corresponds to an S → S and/or an S → S transition, which is present in all photointermediates.

How Photoswitchable Lipids Affect the Order and Dynamics of Lipid Bilayers and Embedded Proteins.

Altering the properties of phospholipid membranes by light is an attractive option for the noninvasive manipulation of membrane proteins and cellular functions. Lipids with an azobenzene group within their acyl chains such as AzoPC are suitable tools for manipulating lipid order and dynamics through a light-induced -to- isomerization. However, the action of these photoswitchable lipids at the atomic level is still poorly understood.

The Desensitized Channelrhodopsin-2 Photointermediate Contains 13 -cis, 15 -syn Retinal Schiff Base.

Channelrhodopsin-2 (ChR2) is a light-gated cation channel and was used to lay the foundations of optogenetics. Its dark state X-ray structure has been determined in 2017 for the wild-type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models.

Real-time nuclear magnetic resonance spectroscopy in the study of biomolecular kinetics and dynamics.

The review describes the application of nuclear magnetic resonance (NMR) spectroscopy to study kinetics of folding, refolding and aggregation of proteins, RNA and DNA. Time-resolved NMR experiments can be conducted in a reversible or an irreversible manner. In particular, irreversible folding experiments pose large requirements for (i) signal-to-noise due to the time limitations and (ii) synchronising of the refolding steps.

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR.

The functional mechanism of the light-driven sodium pump rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13- retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond.

Cysteine oxidation and disulfide formation in the ribosomal exit tunnel.

Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel.

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes.

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant.

The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy.

Light-Induced Uncaging for Time-Resolved Observations of Biochemical Reactions by MAS NMR Spectroscopy.

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E.

Exploring Protein Structures by DNP-Enhanced Methyl Solid-State NMR Spectroscopy.

Although the rapid development of sensitivity-enhanced solid-state NMR (ssNMR) spectroscopy based on dynamic nuclear polarization (DNP) has enabled a broad range of novel applications in material and life sciences, further methodological improvements are needed to unleash the full potential of DNP-ssNMR. Here, a new methyl-based toolkit for exploring protein structures is presented, which combines signal-enhancement by DNP with heteronuclear Overhauser effect (hetNOE), carbon-carbon-spin diffusion (SD) and strategically designed isotope-labeling schemes. It is demonstrated that within this framework, methyl groups can serve as dynamic sensors for probing local molecular packing within proteins.

Opportunities for Successful Stabilization of Poor Glass-Forming Drugs: A Stability-Based Comparison of Mesoporous Silica Versus Hot Melt Extrusion Technologies.

Amorphous formulation technologies to improve oral absorption of poorly soluble active pharmaceutical ingredients (APIs) have become increasingly prevalent. Currently, polymer-based amorphous formulations manufactured by spray drying, hot melt extrusion (HME), or co-precipitation are most common. However, these technologies have challenges in terms of the successful stabilization of poor glass former compounds in the amorphous form.

Incorporation of HPMCAS during loading of glibenclamide onto mesoporous silica improves dissolution and inhibits precipitation.

Mesoporous silica has emerged as an enabling formulation for poorly soluble active pharmaceutical ingredients (APIs). Unlike other formulations, mesoporous silica typically does not inhibit precipitation of supersaturated API therefore, a suitable precipitation inhibitor (PI) should be added to increase absorption from the gastrointestinal (GI) tract. However, there is limited research about optimal processes for combining PIs with silica formulations.