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The x-ray crystal structure of Thermus aquaticus DNA methyltransferase M-Taq I, in complex with S-adenosylmethionine, at 2.4-angstrom resolution exhibits the alpha/beta folding of the polypeptide into two equal sized-regions. The two regions form a C with a wide cleft that can accommodate the DNA substrate. The N-terminal domain is occupied by a nine-standard beta-sheet and the C-terminal domain by four small beta-sheets and alpha-helices. The beta sheet has two conserved segments typical of N-methyltransferases, which form a pocket for cofactor binding. The three-dimensional folding of M-Taq I resembles that of the cytosine-specific Hha I methyltransferase.

An epilimnetic volume of nearly 350,000 m3 was separated by the installation of in overflown, submerged flexible curtain (SFC,160 m long, 6 m deep) perpendicular to the flow direction in the Haselbach mouth region of the Saidenbach Reservoir (Saxony, Germany). The SFC produced (i) an interruption of hydraulic short circuits between inlet and main reservoir basin, (ii) a longer retention time of theinflowing water, and (iii) a 30% to 40% increase of the elimination of soluble reactive phosphorus in the mouth region during summer stratification. It is conclusive that flexible curtains may be cost- effective substitutes for conventional pre-dams of thermally stratified lakes or reservoirs. [ABSTRACT FROM AUTHOR]

The G protein-coupled adenosine A 2A receptor (A 2A AR) is an important new (potential) drug target in immuno-oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A 2A AR antagonists displaying exceptional selectivity. While crystal structures of the human A 2A AR have been solved, mostly using the A 2A -StaR2 protein that bears 9 point mutations, co-crystallization with Preladenant derivatives has so far been elusive. We developed a new A 2A AR construct harboring a single point mutation (S91 3.39 K) which renders it extremely thermostable. This allowed the co-crystallization of two novel Preladenant derivatives, the polyethylene glycol-conjugated (PEGylated) PSB-2113, and the fluorophore-labeled PSB-2115. The obtained crystal structures (2.25 Å and 2.6 Å resolution) provide explanations for the high potency and selectivity of Preladenant derivatives. They represent the first crystal structures of a GPCR in complex with PEG- and fluorophore-conjugated ligands. The applied strategy is predicted to be applicable to further class A GPCRs., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The serotonin transporter belongs to the family of sodium-chloride coupled neurotransmitter transporter and is related to depression in humans. It is therefore an important drug target to support treatment of depression. Recently, structures of human serotonin transporter in complex with inhibitor molecules have been published. However, the production of large protein amounts for crystallization experiments remains a bottleneck. Here we present the possibility to obtain purified serotonin transporter from E. coli. Fos-choline 12 solubilized target protein was obtained with a purity of >95% and a yield of 1.2 mg L -1 culture in autoinduction medium. CD spectroscopic analysis of protein stability allowed identifying CHS and POPX as stabilizing components, which increased hSERT thermostability by 7 °C. The kinetic dissociation constant K D of 2.8 μM (±0.05) for of the inhibitor Desipramine was determined with a ka of 10,848 M - 1 s-1 (±220) and a kd of 0.03 s-1 (±4.7 × 10-5)., (Copyright © 2019. Published by Elsevier Inc.)

Crosstalk between opioid and adrenergic receptors is well characterized and due to interactions between second messenger systems, formation of receptor heterodimers, and extracellular allosteric binding regions. Both classes of receptors bind both sets of ligands. We propose here that receptor crosstalk may be mirrored in ligand complementarity. We demonstrate that opioids bind to adrenergic compounds with micromolar affinities. Additionally, adrenergic compounds bind with micromolar affinities to extracellular loops of opioid receptors while opioids bind to extracellular loops of adrenergic receptors. Thus, each compound type can bind to the complementary receptor, enhancing the activity of the other compound type through an allosteric mechanism. Screening for ligand complementarity may permit the identification of other mutually-enhancing sets of compounds as well as the design of novel combination drugs or tethered compounds with improved duration and specificity of action., Competing Interests: The authors declare no conflict of interest.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

Structure and function studies of membrane proteins, particularly G protein-coupled receptors and multipass transmembrane proteins, require detergents. We have devised a simple tool, the QTY code (glutamine, threonine, and tyrosine), for designing hydrophobic domains to become water soluble without detergents. Here we report using the QTY code to systematically replace the hydrophobic amino acids leucine, valine, isoleucine, and phenylalanine in the seven transmembrane α-helices of CCR5, CXCR4, CCR10, and CXCR7. We show that QTY code-designed chemokine receptor variants retain their thermostabilities, α-helical structures, and ligand-binding activities in buffer and 50% human serum. CCR5 QTY , CXCR4 QTY , and CXCR7 QTY also bind to HIV coat protein gp41-120. Despite substantial transmembrane domain changes, the detergent-free QTY variants maintain stable structures and retain their ligand-binding activities. We believe the QTY code will be useful for designing water-soluble variants of membrane proteins and other water-insoluble aggregated proteins., Competing Interests: Conflict of interest statement: Massachusetts Institute of Technology (MIT) filed several patent applications for the QTY code, and OH2 Laboratories licensed the technology from MIT. This research was funded in part by OH2 Laboratories. S.Z. has a minor equity in OH2 Laboratories.

The data presented here are related to the research article entitled "Expression, purification, and preliminary characterization of human presenilin-2" [1]. Human Presenilin-2 is the catalytic subunit of γ-secretase and a possible calcium leakage channel (Kimberly et al., 2000; Tu et al., 2006) [2], [3]. HisPS2 which was obtained by overexpression in E. coli strain C43 (DE3) was extracted by detergent solubilisation. The sample isolation efficiency by detergents and the protein identification by mass spectrometry and western blot are described. This data article describes the near and far UV circular dichroism measurements and the data deconvolution in terms of secondary structure at 4 and 98 °C. Also, a refolding spectrum is presented. The raw CD spectra used for deconvolution of the helix and stand segments and average length are deposited into Protein Circular Dichroism Data Bank with PCDDBid: CD0005962000 (4 °C far UV), CD0005963000 (98 °C far UV), CD0005964000 (back to 4 °C far UV) and CD0005965000 (4 °C near UV CD).

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists., Competing Interests: The authors declare that they have no conflicts of interest.

Presenilin (PS1 or PS2) functions as the catalytic subunit of γ-secretase, which produces the toxic amyloid beta peptides in Alzheimer's disease (AD). The dependence of folding and structural stability of PSs on the lipophilic environment and mutation were investigated by far UV CD spectroscopy. The secondary structure content and stability of PS2 depended on the lipophilic environment. PS2 undergoes a temperature-dependent structural transition from α-helical to β-structure at 331 K. The restructured protein formed structures which tested positive in spectroscopic amyloid fibrils assays. The AD mutant PS1L266F, PS1L424V and PS1ΔE9 displayed reduced stability which supports a proposed 'loss of function' mechanism of AD based on protein instability. The exon 9 coded sequence in the inhibitory loop of the zymogen was found to be required for the modulation of the thermal stability of PS1 by the lipophilic environment.

Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of Alzheimer's disease patients. We present the structure of an Aβ(1-42) fibril composed of two intertwined protofilaments determined by cryo-electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-β structure resulting in an overall "LS"-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed "groove" and "ridge," leading to different binding pathways on both fibril ends, which has implications for fibril growth., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Par-4 is a unique proapoptotic protein with the ability to induce apoptosis selectively in cancer cells. The X-ray crystal structure of the C-terminal domain of Par-4 (Par-4 CC ), which regulates its apoptotic function, was obtained by MAD phasing. Par-4 homodimerizes by forming a parallel coiled-coil structure. The N-terminal half of Par-4 CC contains the homodimerization subdomain. This structure includes a nuclear export signal (Par-4 NES ) sequence, which is masked upon dimerization indicating a potential mechanism for nuclear localization. The heteromeric-interaction models specifically showed that charge interaction is an important factor in the stability of heteromers of the C-terminal leucine zipper subdomain of Par-4 (Par-4 LZ ). These heteromer models also displayed NES masking capacity and therefore the ability to influence intracellular localization.

Nicastrin, the largest member among the four components of the γ-secretase complex, has been identified to be the substrate recognizer for the proteolytic activity of the complex. Here we report that full-length human nicastrin (hNCT) can be obtained by heterologous expression in E. coli. Milligram quantities of the target protein are purified in a two-step purification protocol using affinity chromatography followed by SEC. The FOS-choline 14 purified tetrameric hNCT exhibits a proper folding with 31% α-helix and 23% β-sheet content. Thermal stability studies reveal stable secondary and tertiary structure of the detergent purified hNCT. A physical interaction between nicastrin and the γ-secretase substrate APPC100 confirmed the functionality of hNCT as a substrate recognizer.

It has long been known in spectroscopy that light not passing through a sample, but reaching the detector (i.e., stray light), results in a distortion of the spectrum known as absorption flattening. In spectroscopy with crystals, one must either include such stray light or take steps to exclude it. In the former case, the derived spectra are not accurate. In the latter case, a significant amount of the crystal must be masked off and excluded. In this paper, we describe a method that allows use of the entire crystal by correcting the distorted spectrum.

Pyruvate dehydrogenase and oxoglutarate dehydrogenase catalyze key reactions in central metabolism. In Corynebacterium glutamicum and related bacteria like Mycobacterium tuberculosis both activities reside in a novel protein supercomplex with the fusion protein OdhA catalyzing the conversion of oxoglutarate to succinyl-coenzyme A. This activity is inhibited by the forkhead-associated (FHA) domain of the small autoinhibitory protein OdhI. Here we used a biological screen which enabled us to isolate suppressor mutants that are influenced in OdhA-OdhI interaction. Five mutants carrying an OdhI mutation were isolated and one with an OdhA mutation. The OdhA mutein OdhA-C704E and three additional C704 variants were constructed. They exhibited unaltered or even slightly enhanced OdhA activity but showed reduced inhibition and interaction with OdhI. The FHA domain of OdhI was crystallized and its structure found in full agreement with previously determined NMR structures. Based on further structural studies, OdhA-OdhI crosslinking experiments, and modeling we discuss the experimental data generated on OdhA-OdhI interaction, with the latter protein representing a rare example of an FHA domain also recognizing a non-phosphorylated interaction partner., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteins via its C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to be P41212. The unit-cell parameters are a = b = 115.351, c = 123.663 Å, α = β = γ = 90°.

Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended., (© 2014 Elsevier Inc. All rights reserved.)

N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D) of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

The presented method to crystallize membrane proteins combines the advantages of the meso-phase crystallization method and the classical vapor diffusion crystallization. It allows fast screening of crystallization conditions employing automated liquid handlers suited for the 96-well crystallization format.

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time.

This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of immunoglobulins and the Chelex method. The most relevant application- purification of histidine-tagged recombinant proteins-will be reviewed in greater detail with focus of state-of-the-art materials, methods, and protocols, and the limitations of IMAC and recent advances to improve the technology and the methods will be described., (Copyright © 2011. Published by Elsevier Inc.)

Sensory rhodopsin I (SRI) from Halobacterium salinarum mediates both positive and negative phototaxis in a light-dependent manner. SRI photoactivation elicits extensive structural changes which are transmitted to the cognate transducer (HtrI). The atomic structure of the SRI-HtrI complex has not been solved yet and, therefore, details on the interaction which define the binding site between receptor and transducer are missing. The related complex SRII-HtrII from Natronobacterium pharaonis exhibits a hydrogen bond between the receptor Y199 and transducer N54. This bond has been suggested to mediate signal relay in the SRII-HtrII system. Our previous results on the SRI-HtrI complex indicated that HtrI N53 forms a hydrogen bond at the cytoplasm-proximity of the membrane. Here, based on kinetic and spectroscopic data, we demonstrate that Y210 of SRI is functionally significant for the signal relay in the SRI-HtrI complex. Each of the tyrosine residues Y197, Y208, Y210 and Y213 were conservatively exchanged for phenylalanine but only the Y210F mutation led to the disappearance of the infrared band of the terminal amide C=O of N53. From this FT-IR spectroscopic result, we conclude that Y210 of SRI and N53 of HtrI interact via a hydrogen bond which is crucial for the signal transfer from the light receptor to the transducer.

This article reviews the development of immobilized-metal affinity chromatography (IMAC) and describes its most important applications. We provide an overview on the use of IMAC in protein fractionation and proteomics, in protein immobilization and detection, and on some special applications such as purification of immunoglobulins and the Chelex method. The most relevant application-purification of histidine-tagged recombinant proteins-will be reviewed in greater detail with focus of state-of-the-art materials, methods, and protocols, and the limitations of IMAC and recent advances to improve the technology and the methods will be described.

We describe an efficient strategy to produce high-quality proteins by using a single large IMAC chromatography column and enzymatic His-tag removal via the TAGZyme system in pilot scale. Numerous quality assays demonstrated a high purity of the final product, the human cytokine Interleukin-1beta (IL-1beta). The protein preparation was apparently free of host cell proteins, endotoxins, protease, and aggregates. The N-terminal amino acid sequence of IL-1beta was in full agreement with the natural mature form of IL-1beta. The homogeneity of the product was further shown by X-ray structure determination which confirmed the previously solved structure of the protein. We propose the applied workflow as a strategy for industrial production of protein-based biopharmaceuticals.

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15 degrees of helix TM2 and a displacement of this helix by 0.9 A at the cytoplasmic surface.

Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer complex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the receptor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mechanism might also be relevant for eubacterial chemoreceptor signalling.