ABSTRACT
The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of Gq proteins by blocking GDP/GTP exchange. The inhibitor binding site of Gq 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. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects.
ABSTRACT
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. They collectively explain the observed color shift. Furthermore, analysis of the differences in chemical shift between nuclei within the same residues in green and blue proteorhodopsins also reveals a correlation with the respective degree of conservation. Our data show that the highly conserved color change mainly affects other highly conserved residues, illustrating a high degree of robustness of the color phenotype to sequence variation.
Subject(s)
Biological Evolution , Cell Nucleus , Rhodopsins, Microbial , Glutamine , NorisoprenoidsABSTRACT
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. Previous studies suggested the involvement of two intracellular loops called coupling helix 1 and 2 (CH1, CH2). Here, we demonstrate by solid-state NMR spectroscopy that substantial chemical shift changes within both CH1 and CH2 occur upon substrate binding, in the ATP hydrolysis transition state, and upon inhibitor binding. CH2 is domain-swapped within the MsbA structure, and it is noteworthy that substrate binding induces a larger response in CH2 compared to CH1. Our data demonstrate that CH1 and CH2 undergo structural changes as part of the TMD-NBD cross-talk.
Subject(s)
ATP-Binding Cassette Transporters , Magnetic Resonance Imaging , Cross Reactions , Magnetic Resonance Spectroscopy , Adenosine TriphosphateABSTRACT
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 15N and 13C of membrane proteins. The convergence of the AF-QM/MM method was tested using Krokinobacter eikastus rhodopsin 2 as a test case. When the distance threshold of the QM region is equal to or larger than 4.0 Å, the results of the AF-QM/MM calculations are close to convergence. In addition, the effects of selected density functionals, basis sets, and local chemical environment of target atoms on the chemical shift calculations were systematically investigated. Our results demonstrate that the predicted chemical shifts are more accurate when important environmental factors including cross-protomer interactions, lipid molecules, and solvent molecules are taken into consideration, especially for the 15N chemical shift prediction. Furthermore, with the presence of sodium ions in the environment, the chemical shift of residues, retinal, and retinal Schiff base are affected, which is consistent with the results of the solid-state nuclear magnetic resonance (NMR) experiment. Upon comparing the performance of various density functionals (namely, B3LYP, B3PW91, M06-2X, M06-L, mPW1PW91, OB95, and OPBE), the results show that mPW1PW91 is a suitable functional for the 15N and 13C chemical shift prediction of the membrane proteins. Meanwhile, we find that the improved accuracy of the 13Cß chemical shift calculations can be achieved by the employment of the triple-ζ basis set. However, the employment of the triple-ζ basis set does not improve the accuracy of the 15N and 13Cα chemical shift calculations nor does the addition of a diffuse function improve the overall prediction accuracy of the chemical shifts. Our study also underscores that the AF-QM/MM method has significant advantages in predicting the chemical shifts of key ligands and nonstandard residues in membrane proteins than most widely used empirical models; therefore, it could be an accurate computational tool for chemical shift calculations on various types of biological systems.
Subject(s)
Membrane Proteins , Molecular Dynamics Simulation , Magnetic Resonance Spectroscopy , Rhodopsin , Solvents/chemistry , Quantum TheoryABSTRACT
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. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.
ABSTRACT
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. We herein present the synthesis and photophysical characterization of a 2P-sensitive uncaging dyad based on rhodamine 101 as donor fluorophore and a redshifted BODIPY as acceptor photocage. Liberation of p-nitroaniline (PNA) upon one-photon photolysis was confirmed by HPLC analysis. The photoreaction was found to be accompanied by a considerable change of the fluorescence properties of the chromophores. The possibility of a fluorescent read-out enabled the detection of two-photon induced uncaging by confocal fluorescence microscopy.
ABSTRACT
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. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.
Subject(s)
Rhodopsin , Sodium-Potassium-Exchanging ATPase , Sodium-Potassium-Exchanging ATPase/metabolism , Rhodopsin/chemistry , Models, Molecular , Mutation , Sodium/metabolism , LightABSTRACT
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. Important insight can be gained by solid-state NMR spectroscopy by which the large homo-oligomeric rhodopsin complexes can be studied directly within lipid bilayers. This chapter describes the methodological background and the necessary sample preparation requirements for the study of photointermediates, for the analysis of protonation states, H-bonding and chromophore conformations, for 3D structure determination, and for probing oligomer interfaces of microbial rhodopsins. The use of data extracted from these NMR experiments is discussed in the context of complementary biophysical methods.
Subject(s)
Rhodopsin , Rhodopsins, Microbial , Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy/methods , Molecular Conformation , Rhodopsin/chemistry , Rhodopsins, Microbial/chemistryABSTRACT
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. This so-called precipitate-forming cell-free (P-CF) expression mode is a very effective and reliable approach for numerous applications. Even from complex membrane proteins such as G-protein coupled receptors or large transporters, significant amounts of such precipitates can be synthesized within few hours. The precipitates can be solubilized in detergents or reconstituted into membranes for subsequent structural or functional analysis. Harsh denaturation and refolding procedures as known from the treatment of bacterial inclusion bodies are usually not required.This strategy is particularly interesting for applications requiring large amounts of membrane protein or fast access to a sample. It is further an excellent tool for the production of membrane protein antigens suitable for antibody generation. The purification of the precipitates in downstream processing is streamlined as only few proteins from the cell-free lysate may co-precipitate with the synthesized membrane protein. For most applications, a one-step affinity chromatography by taking advantage of small purification tags attached to the membrane protein target is sufficient. We give an overview on current applications of P-CF precipitates and describe the underlying techniques in detail. We furthermore provide protocols for the successful crystallization and NMR analysis of P-CF synthesized membrane proteins exemplified with the diacylglycerol kinase (DAGK). In addition, we describe the functional characterization of a P-CF synthesized large eukaryotic transporter.
Subject(s)
Membrane Proteins , Protein Folding , Cell-Free System/metabolism , Detergents/metabolism , Escherichia coli/metabolism , Membrane Proteins/metabolism , Protein Processing, Post-TranslationalABSTRACT
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. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Biological Transport , Electron Spin Resonance Spectroscopy/methods , Lipopolysaccharides/metabolismABSTRACT
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. In liquid-state this revolutionizing technique has been restricted to a few specific non-biological model molecules in organic solvents. Here we show that the carbon polarization in small biological molecules, including carbohydrates and amino acids, can be enhanced sizably by in situ Overhauser DNP (ODNP) in water at room temperature and at high magnetic field. An observed connection between ODNP 13C enhancement factor and paramagnetic 13C NMR shift has led to the exploration of biologically relevant heterocyclic compound indole. The QM/MM MD simulation underscores the dynamics of intermolecular hydrogen bonds as the driving force for the scalar ODNP in a long-living radical-substrate complex. Our work reconciles results obtained by DNP spectroscopy, paramagnetic NMR and computational chemistry and provides new mechanistic insights into the high-field scalar ODNP.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Water/chemistry , Carbon Isotopes/chemistry , Density Functional Theory , Hydrogen Bonding , Magnetic Fields , Molecular Dynamics Simulation , TemperatureABSTRACT
We report a transient signature in the near-UV absorption of Krokinobacter eikastus rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all-trans to 13-cis isomerization of retinal and decays with the reisomerization to all-trans 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 S0 â S3 and/or an S0 â S5 transition, which is present in all photointermediates. These transitions exhibit a negligible spectral shift by the altering protein environment, in contrast to the main absorption band. This is rationalized by the extension of the transition densities that omits the Schiff base nitrogen. Further characterization and first steps into possible optogenetic applications were performed with near-UV quenching experiments of an induced photostationary state, yielding an ultrafast regeneration of the parent state of KR2.
Subject(s)
Absorption, Physicochemical , Flavobacteriaceae/metabolism , Rhodopsin/chemistry , Rhodopsin/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Ultraviolet Rays , Cell Membrane/metabolism , Flavobacteriaceae/cytology , Molecular Dynamics Simulation , Protein Conformation , Spectrum AnalysisABSTRACT
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 trans-to-cis isomerization. However, the action of these photoswitchable lipids at the atomic level is still poorly understood. Here, liposomes containing AzoPC, POPE, and POPG have been characterized by solid-state NMR through chemical shift and dipolar CH order parameter measurements. Upon UV-light illumination, an efficient trans-to-cis conversion can be achieved resulting in a localized reduction of the CH order parameter within the bulk lipid acyl chains. This effect is even more pronounced in liposomes containing the integral membrane protein E. coli diacylglycerol kinase. The protein responds to the light-induced trans-to-cis isomerization by a site-specific increase in the molecular dynamics as observed by altered cross peak intensities in NCA spectra. This study represents a proof-of-concept demonstration for the use of photoswitchable lipids to modulate membrane properties by light for inducing dynamic changes within an embedded membrane protein.
ABSTRACT
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. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP-enhanced solid-state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle.
Subject(s)
Channelrhodopsins/chemistry , Chlamydomonas reinhardtii/chemistry , Diterpenes/chemistry , Retinaldehyde/chemistry , Schiff Bases/chemistry , Cations/chemistry , Light , Magnetic Resonance Spectroscopy , Photochemical Processes , Photons , Protein ConformationABSTRACT
The functional mechanism of the light-driven sodium pump Krokinobacter eikastus 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-cis 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. The retinal converts back into an all-trans conformation in the O-intermediate, which is the key state for sodium transport. However, retinal carbon and Schiff base nitrogen chemical shifts differ from those observed in the KR2 dark state all-trans conformation, indicating a perturbation through the nearby bound sodium ion. Our findings are supplemented by optical and infrared spectroscopy and are discussed in the context of known three-dimensional structures.
Subject(s)
Rhodopsin , Sodium-Potassium-Exchanging ATPase , Carbon/metabolism , Flavobacteriaceae , Ions/metabolism , Magnetic Resonance Spectroscopy , Rhodopsin/chemistry , Sodium/chemistry , Sodium-Potassium-Exchanging ATPase/chemistryABSTRACT
Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. Here, we present a well-defined homo-trimeric structure of DgkA in phospholipid bilayers determined by magic angle spinning solid-state NMR (ssNMR) spectroscopy, using an approach combining intra-, inter-molecular paramagnetic relaxation enhancement (PRE)-derived distance restraints and CS-Rosetta calculations. The DgkA structure determined in lipid bilayers is different from the solution NMR structure. In addition, although ssNMR structure of DgkA shows a global folding similar to that determined by X-ray, these two structures differ in monomeric symmetry and dynamics. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins.
Subject(s)
Diacylglycerol Kinase/metabolism , Lipid Bilayers/metabolism , Phospholipids/metabolism , Detergents/chemistry , Diacylglycerol Kinase/chemistry , Diacylglycerol Kinase/genetics , Lipid Bilayers/chemistry , Models, Molecular , Mutation , Nuclear Magnetic Resonance, Biomolecular , Phospholipids/chemistry , Protein Conformation , Protein Folding , Protein Multimerization , Structure-Activity RelationshipABSTRACT
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. Thus, this contribution discusses the application of methods for signal-to-noise increases, including dynamic nuclear polarisation, hyperpolarisation and photo-CIDNP for the study of time-resolved NMR studies. Further, methods are reviewed ranging from pressure and temperature jump, light induction to rapid mixing to induce rapidly non-equilibrium conditions required to initiate folding.
ABSTRACT
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. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.
Subject(s)
Cysteine/chemistry , Disulfides/chemistry , Protein Biosynthesis , Ribosomes/chemistry , Ribosomes/metabolism , gamma-Crystallins/chemistry , Cryoelectron Microscopy , Cysteine/metabolism , Glutathione/analogs & derivatives , Glutathione/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Models, Molecular , Mutation , Oxidation-Reduction , Protein Conformation , Protein Folding , Ribosomes/genetics , S-Nitrosothiols/chemistryABSTRACT
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. To study conformational transitions between these states, all six tryptophans of Y2R were 13 C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13 Câ NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp11623.50 was identified.
Subject(s)
Receptors, Neuropeptide Y/chemistry , Humans , Models, Molecular , Molecular ConformationABSTRACT
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. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.