Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the Cα-helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 µs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II.

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.

In cell Gd3+-based site-directed spin labeling and EPR spectroscopy of eGFP.

Label-based functional studies of biomolecules in their native environment require labeling reactions inside living cells. In cell spin labeling using alkyne-azide click chemistry with a Gd3+-DOTAM-azide complex is shown to provide high spin label stability and narrow EPR lines for EPR spectroscopic detection of a spin labeled protein in living cells at ambient temperatures.

Styrene/Maleic Acid Copolymers Form SMALPs by Pulling Lipid Patches out of the Lipid Bilayer.

Amphiphilic copolymers composed of styrene and maleic acid (SMA) monomers caused a major methodical breakthrough in the study of membrane proteins. They were found to directly release phospholipids and membrane proteins both from artificial and natural lipid bilayers, yielding stable water-soluble discoidal SMA/lipid particles (SMALPs) of uniform size. Although many empirical studies indicate the great potency of SMALPs for membrane protein research, the mechanisms of their formation remain obscure. It is unknown which factors account for the very assembly of SMALPs and govern their uniform size. We have developed a coarse-grained (CG) molecular model of SMA copolymers based on the MARTINI CG force field and used it to probe the behavior of SMA copolymers with varying composition/charge/concentration in solution as well as their interaction with lipid membranes. First, we found that SMA copolymers tend to aggregate in solution into clusters, which could account for the uniform size of SMALPs. Next, molecular dynamics (MD) simulations showed that periodic SMA copolymers with styrene/maleic acid ratios of 2:1 ([SSM] n) and 3:1 ([SSSM] n) differently interacted with lipid bilayers. While clusters of 2:1 SMA copolymers induced membrane poration, the clusters of 3:1 SMA copolymers extracted lipid patches from the membrane yielding SMALP-like structures. Extraction of lipid patches was also observed when we simulated the behavior of 3:1 copolymers with varying lengths and statistical distribution of styrene and MA units. Analysis of MD simulation trajectories and comparison with experimental data indicate that the formation of SMALPs requires copolymer molecules with a sufficient number of units made of more than two sequential styrene monomers.

Relationship of Catalysis and Active Site Loop Dynamics in the (ßα)8-Barrel Enzyme Indole-3-glycerol Phosphate Synthase.

It is important to understand how the catalytic activity of enzymes is related to their conformational flexibility. We have studied this activity-flexibility correlation using the example of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (ssIGPS), which catalyzes the fifth step in the biosynthesis of tryptophan. ssIGPS is a thermostable representative of enzymes with the frequently encountered and catalytically versatile (ßα)8-barrel fold. Four variants of ssIGPS with increased catalytic turnover numbers were analyzed by transient kinetics at 25 °C, and wild-type ssIGPS was likewise analyzed both at 25 °C and at 60 °C. Global fitting with a minimal three-step model provided the individual rate constants for substrate binding, chemical transformation, and product release. The results showed that in both cases, namely, the application of activating mutations and temperature increase, the net increase in the catalytic turnover number is afforded by acceleration of the product release rate relative to the chemical transformation steps. Measurements of the solvent viscosity effect at 25 °C versus 60 °C confirmed this change in the rate-determining step with temperature, which is in accordance with a kink in the Arrhenius diagram of ssIGPS at ∼40 °C. When rotational diffusion rates of electron paramagnetic spin-labels attached to active site loop ß1α1 are plotted in the form of an Arrhenius diagram, kinks are observed at the same temperature. These findings, together with molecular dynamics simulations, demonstrate that a different degree of loop mobility correlates with different rate-limiting steps in the catalytic mechanism of ssIGPS.

Conformational Changes and Competitive Adsorption between Serum Albumin and Hemoglobin on Bioceramic Substrates.

Traditional methods to analyze interactions and conformational changes of proteins adsorbed onto biomaterials are limited by the protein's associations with the substrate material and the complexity of the surrounding media. We have used EPR spectroscopy in combination with site-directed spin labeling (SDSL) to investigate single protein and competitive adsorption kinetics of horse hemoglobin (Hgb) and bovine serum albumin (BSA) on a silica-calcium-phosphate bioceramic substrate. Combined continuous wave and pulsed (DEER) EPR techniques were employed to monitor local mobility/flexibility changes within the proteins and tertiary structure dynamics upon adsorption. An alternate labeling technique was introduced to allow for specific quantification of each protein adsorbed to the bioceramic surface. We show that at buffer pH 7.4 and 4.7 the amount of adsorbed hemoglobin was increased by a factor of 4-5 compared with BSA. The tertiary structure of hemoglobin was strongly affected upon adsorption, leading to a dissociation of the tetrameric molecule into monomers or αß dimers. When the bioceramic substrate was previously functionalized with a layer of BSA, dissociation was reduced by 71 % compared with the untreated surface, indicating a "primer" effect of BSA for better adhesion of the globular hemoglobin.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of Natronomonas pharaonis.

Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

Clustering and dynamics of phototransducer signaling domains revealed by site-directed spin labeling electron paramagnetic resonance on SRII/HtrII in membranes and nanodiscs.

In halophilic archaea the photophobic response is mediated by the membrane-embedded 2:2 photoreceptor/-transducer complex SRII/HtrII, the latter being homologous to the bacterial chemoreceptors. Both systems bias the rotation direction of the flagellar motor via a two-component system coupled to an extended cytoplasmic signaling domain formed by a four helical antiparallel coiled-coil structure. For signal propagation by the HAMP domains connecting the transmembrane and cytoplasmic domains, it was suggested that a two-state thermodynamic equilibrium found for the first HAMP domain in NpSRII/NpHtrII is shifted upon activation, yet signal propagation along the coiled-coil transducer remains largely elusive, including the activation mechanism of the coupled kinase CheA. We investigated the dynamic and structural properties of the cytoplasmic tip domain of NpHtrII in terms of signal transduction and putative oligomerization using site-directed spin labeling electron paramagnetic resonance spectroscopy. We show that the cytoplasmic tip domain of NpHtrII is engaged in a two-state equilibrium between a dynamic and a compact conformation like what was found for the first HAMP domain, thus strengthening the assumption that dynamics are the language of signal transfer. Interspin distance measurements in membranes and on isolated 2:2 photoreceptor/transducer complexes in nanolipoprotein particles provide evidence that archaeal photoreceptor/-transducer complexes analogous to chemoreceptors form trimers-of-dimers or higher-order assemblies even in the absence of the cytoplasmic components CheA and CheW, underlining conservation of the overall mechanistic principles underlying archaeal phototaxis and bacterial chemotaxis systems. Furthermore, our results revealed a significant influence of the NpHtrII signaling domain on the NpSRII photocycle kinetics, providing evidence for a conformational coupling of SRII and HtrII in these complexes.

Conformational changes of the histidine ATP-binding cassette transporter studied by double electron-electron resonance spectroscopy.

The conformational dynamics of the histidine ABC transporter HisQMP2 from Salmonella enterica serovar Typhimurium, reconstituted into liposomes, is studied by site-directed spin labeling and double electron-electron resonance spectroscopy in the absence of nucleotides, in the ATP-bound, and in the post-hydrolysis state. The results show that the inter-dimer distances as measured between the Q-loops of HisP2 in the intact transporter resemble those determined for the maltose transporter in all three states of the hydrolysis cycle. Only in the presence of liganded HisJ the closed conformation of the nucleotide binding sites is achieved revealing the transmembrane communication of the presence of substrate. Two conformational states can be distinguished for the periplasmic moiety of HisQMP2 as detected by differences in distributions of interspin distances between positions 86 and 96 or 104 and 197. The observed conformational changes are correlated to proposed open, semi-open and closed conformations of the nucleotide binding domains HisP2. Our results are in line with a rearrangement of transmembrane helices 4 and 4' of HisQM during the closed to the semi-open transition of HisP2 driven by the reorientation of the coupled helices 3a and 3b to occur upon hydrolysis.

Domain movements during CCA-addition: a new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases.

CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3'-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3'-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response.

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations.

A high resolution electro-optical approach for investigating transition of soluble proteins to integral membrane proteins probed by colicin A.

The transition from water soluble state to an integral membrane protein state is a crucial step in the formation of the active form of many pore-forming or receptor proteins. Albeit this, high resolution techniques which allow assay of protein membrane binding and concomitant development of the final active form in the membrane await further development. Here, we describe a horizontal artificial bilayers setup allowing for simultaneous electrical and optical measurements at a single molecule level. We use the membrane binding and subsequent channel formation of colicin A (ColA) a water soluble bacteriocin secreted by some strains of Escherichia coli to demonstrate the potential of the combined electro-optical technique. Our results expand the knowledge on ColA molecular details which show that active ColA is monomeric; membrane binding is pH but not membrane-potential (Δϕ) dependent. ColA is at Δϕ=0 permeable for molecules ≥1 nm. Although ColA exhibits low ion conductance it facilitates permeation of large molecules. Our electro-optical recordings reveal ColA monomeric state and the chimeric character of its pore.

Kissing G domains of MnmE monitored by X-ray crystallography and pulse electron paramagnetic resonance spectroscopy.

MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF(x) structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 A. With GDP and AlF(x), which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF(x) requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.

An electron paramagnetic resonance spectroscopic investigation on the growth mechanism of NaYF4:Gd nanocrystals.

Doped nanocrystals of NaYF(4) and NaGdF(4) are currently studied as upconversion luminescence markers and magnetic resonance imaging contrast agents. An EPR investigation on the growth mechanism of NaYF(4):Gd and NaGdF(4) nanocrystals showed that these nanomaterials grow in the standard oleic acid-based reaction medium by a dissolution/recrystallization mechanism and not by the aggregation or oriented attachment of smaller particles.

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review.

Amphiphilic copolymers consisting of alternating hydrophilic and hydrophobic units account for a major recent methodical breakthrough in the investigations of membrane proteins. Styrene-maleic acid (SMA), diisobutylene-maleic acid (DIBMA), and related copolymers have been shown to extract membrane proteins directly from lipid membranes without the need for classical detergents. Within the particular experimental setup, they form disc-shaped nanoparticles with a narrow size distribution, which serve as a suitable platform for diverse kinds of spectroscopy and other biophysical techniques that require relatively small, homogeneous, water-soluble particles of separate membrane proteins in their native lipid environment. In recent years, copolymer-encased nanolipoparticles have been proven as suitable protein carriers for various structural biology applications, including cryo-electron microscopy (cryo-EM), small-angle scattering, and conventional and single-molecule X-ray diffraction experiments. Here, we review the current understanding of how such nanolipoparticles are formed and organized at the molecular level with an emphasis on their chemical diversity and factors affecting their size and solubilization efficiency.

The signal transfer from the receptor NpSRII to the transducer NpHtrII is not hampered by the D75N mutation.

Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.

Monomeric and dimeric conformation of the vinculin tail five-helix bundle in solution studied by EPR spectroscopy.

The cytoskeletal adaptor protein vinculin plays an important role in the control of cell adhesion and migration, linking the actin cytoskeleton to adhesion receptor complexes in cell adhesion sites. The conformation of the vinculin tail dimer, which is crucial for protein function, was analyzed using site-directed spin labeling in electron paramagnetic resonance spectroscopy. Interspin distances for a set of six singly and four doubly spin-labeled mutants of the tail domain of vinculin were determined and used as constraints for modeling of the vinculin tail dimer. A comparison of the results obtained by molecular dynamic simulations and a rotamer library approach reveals that the crystal structure of the vinculin tail monomer is essentially preserved in aqueous solution. The orientation of monomers within the dimer observed previously by x-ray crystallography agrees with the solution electron paramagnetic resonance data. Furthermore, the distance between positions 1033 is shown to increase by >3 nm upon interaction of the vinculin tail domain with F-actin.

Microtubule binding and trapping at the tip of neurites regulate tau motion in living neurons.

During the development of neurons, the microtubule-associated tau proteins show a graded proximo-distal distribution in axons. In tauopathies such as Alzheimer's disease, tau accumulates in the somatodendritic compartment. To scrutinize the determinants of tau's distribution and motion, we constructed photoactivatable green fluorescent protein (GFP)-tagged tau fusion proteins and recorded their distribution after focal activation in living cells. Simulation showed that the motion of tau was compatible with diffusion/reaction as opposed to active transport/reaction. Effective diffusion constants of 0.7-0.8 microm(2)/second were calculated in neurites of PC12 cells and primary cortical neurons. Furthermore, tau's amino terminal projection domain mediated binding and enrichment of tau at distal neurites indicating that the tip of a neurite acts as an adsorber trapping tau protein. Treatment with taxol, incorporation of disease-related tau modifications, experimentally induced hyperphosphorylation and addition of preaggregated amyloid beta peptides (Abeta) increased the effective diffusion constant compatible with a decreased binding to microtubules. Distal enrichment was present after taxol treatment but was suppressed at disease-relevant conditions. The data suggest that (i) dynamic binding of tau to microtubules and diffusion along microtubules and (ii) trapping at the tip of a neurite both contribute to its distribution during development and disease.