Sina Reckel

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems.

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples. The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain. Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient. This protocol describes the preparation of all essential reaction components such as the E. coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions. The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated. We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h.

Large-scale production of functional membrane proteins

Abstract.The preparation of sufficient amounts of high-quality samples is still the major bottleneck for the characterization of membrane proteins by in vitro approaches. The hydrophobic nature, the requirement for complicated transport and modification pathways, and the often observed negative effects on membrane properties are intrinsic features of membrane proteins that frequently cause significant problems in overexpression studies. Establishing efficient protocols for the production of functionally folded membrane proteins is therefore a challenging task, and numerous specific characteristics have to be considered. In addition, a variety of expression systems have been developed, and choice of appropriate techniques could strongly depend on the desired target membrane proteins as well as on their intended applications. The production of membrane proteins is a highly dynamic field and new or modified approaches are frequently emerging. The review will give an overview of currently established processes for the production of functionally folded membrane proteins.

Solution NMR structure of proteorhodopsin.

and we show herein the de novo structure ofthegreenvariantofproteorhodopsinsolvedbysolutionNMRspectroscopy.The structure of PR (Figure 1) was solved in the short-chain lipid diC7PC (diheptanoyl-phosphocholine) combininglong-range NOEs with restraints derived from paramagneticrelaxation enhancement (PRE) and residual dipolar cou-plings (RDCs). The seven transmembrane helices are con-nected by short loops. Instead of the anti-parallel b-sheet thatis observed between helices B and C in other microbialrhodopsins, torsion angles derived from the protein backbonedihedral angle prediction program TALOS+ suggest that PRresidues G87–P90 form a short b-turn. The loop betweenhelices D and E is longer than predicted by the secondarystructure prediction program TMHMM.

Cell-free expression and stable isotope labelling strategies for membrane proteins.

Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on α-helical transmembrane proteins which benefit most from such methods.

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense

BACKGROUND AND PURPOSE Frankincense, the gum resin derived from Boswellia species, showed anti‐inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti‐inflammatory efficacy in vivo and their molecular modes of action are incompletely understood.

The Molecular Pharmacology and In Vivo Activity of 2-(4-Chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid (YS121), a Dual Inhibitor of Microsomal Prostaglandin E2 Synthase-1 and 5-Lipoxygenase

The microsomal prostaglandin E2 synthase (mPGES)-1 is one of the terminal isoenzymes of prostaglandin (PG) E2 biosynthesis. Pharmacological inhibitors of mPGES-1 are proposed as an alternative to nonsteroidal anti-inflammatory drugs. We recently presented the design and synthesis of a series of pirinixic acid derivatives that dually inhibit mPGES-1 and 5-lipoxygenase. Here, we investigated the mechanism of mPGES-1 inhibition, the selectivity profile, and the in vivo activity of α-(n-hexyl)-substituted pirinixic acid [YS121; 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid)] as a lead compound. In cell-free assays, YS121 inhibited human mPGES-1 in a reversible and noncompetitive manner (IC50 = 3.4 μM), and surface plasmon resonance spectroscopy studies using purified in vitro-translated human mPGES-1 indicate direct, reversible, and specific binding to mPGES-1 (KD = 10–14 μM). In lipopolysaccharide-stimulated human whole blood, PGE2 formation was concentration dependently inhibited (IC50 = 2 μM), whereas concomitant generation of the cyclooxygenase (COX)-2-derived thromboxane B2 and 6-keto PGF1α and the COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was not significantly reduced. In carrageenan-induced rat pleurisy, YS121 (1.5 mg/kg i.p.) blocked exudate formation and leukocyte infiltration accompanied by reduced pleural levels of PGE2 and leukotriene B4 but also of 6-keto PGF1α. Taken together, these results indicate that YS121 is a promising inhibitor of mPGES-1 with anti-inflammatory efficiency in human whole blood as well as in vivo.

Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm.

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with 13C or/and 15N such that cross peaks between 13CO(i – 1) and 15NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with 13C and the second with 15N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B2R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.

Strategies for the cell-free expression of membrane proteins.

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.

In-cell solid-state NMR as a tool to study proteins in large complexes.

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.

Transmembrane segment enhanced labeling as a tool for the backbone assignment of α-helical membrane proteins

Recent advances in cell-free expression protocols have opened a new avenue toward high-resolution structural investigations of membrane proteins by x-ray crystallography and NMR spectroscopy. One of the biggest challenges for liquid-state NMR-based structural investigations of membrane proteins is the significant peak overlap in the spectra caused by large line widths and limited chemical shift dispersion of α-helical proteins. Contributing to the limited chemical shift dispersion is the fact that ≈60% of the amino acids in transmembrane regions consist of only six different amino acid types. This principle disadvantage, however, can be exploited to aid in the assignment of the backbone resonances of membrane proteins; by 15N/13C-double-labeling of these six amino acid types, sequential connectivities can be obtained for large stretches of the transmembrane segments where number and length of stretches consisting exclusively of these six amino acid types are enhanced compared with the remainder of the protein. We show by experiment as well as by statistical analysis that this labeling scheme provides a large number of sequential connectivities in transmembrane regions and thus constitutes a tool for the efficient assignment of membrane protein backbone resonances.