Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (2H/15N/13C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding KD values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineerin

Magnetic anisotropy of epitaxial (Ga,Mn)As on (113)A GaAs

The temperature dependence of magnetic anisotropy in (113)A (Ga,Mn)As layers grown by molecular beam epitaxy is studied by means of superconducting quantum interference device (SQUID) magnetometry as well as by ferromagnetic resonance (FMR) and magnetooptical effects. Experimental results are described considering cubic and two kinds of uniaxial magnetic anisotropy. The magnitude of cubic and uniaxial anisotropy constants is found to be proportional to the fourth and second power of saturation magnetization, respectively. Similarly to the case of (001) samples, the spin reorientation transition from uniaxial anisotropy with the easy along the [-1, 1, 0] direction at high temperatures to the biaxial anisotropy at low temperatures is observed around 25 K. The determined values of the anisotropy constants have been confirmed by FMR studies. As evidenced by investigations of the polar magnetooptical Kerr effect, the particular combination of magnetic anisotropies allows the out-of-plane component of magnetization to be reversed by an in-plane magnetic field. Theoretical calculations within the p-d Zener model explain the magnitude of the out-of-plane uniaxial anisotropy constant caused by epitaxial strain, but do not explain satisfactorily the cubic anisotropy constant. At the same time the findings point to the presence of an additional uniaxial anisotropy of unknown origin. Similarly to the case of (001) films, this additional anisotropy can be explained by assuming the existence of a shear strain. However, in contrast to the (001) samples, this additional strain has an out-of-the-(001)-plane character.Comment: 13 pages, 9 figure

Object-Oriented Build Automation – A Case Study

Fast and precise build and deployment automation is a fundamental task for every project oriented on rapidly appearing changes. As a rule of thumb, the tools used for this task work as procedural-declarative frameworks – often overlooking the extra requirements for large projects like easy parallelization, precise targeting of specific subsystem or general code readability. In this article we document our findings in build automation as we have abandoned the procedural-declarative approach to object-oriented perspective of our setup environment – all implemented in the .NET build automation framework Cake Frosting. Due to the clear separation of the various layers of our system and our codebase we are able to fire up our new build-deployment routines at ease and at specific part of our ecosystem. As the whole routine is written as a C# console application we can easily manage some aspects of parallel execution (i.e. number of threads) of some tasks which results in great drop of job execution time. To further improve the execution time, we introduce the concept of proof-of-work which is a file that stores the information about the last successful build. Together, all of our concepts resulted in a fast build-deployment routine – as in pessimistic scenario we managed to drop to about 30 % of the original time. We believe that others may benefit from our case study as the concepts proposed here can be easily incorporated to any other project written in .NET (or one that is built using object-oriented command-line application) – though we would not recommend using our approach in small projects (in terms of KLOC)

Structural and paramagnetic properties of dilute Ga1-xMnxN

Systematic investigations of the structural and magnetic properties of single crystal (Ga,Mn)N films grown by metal organic vapor phase epitaxy are presented. High resolution transmission electron microscopy, synchrotron x-ray diffraction, and extended x-ray absorption fine structure studies do not reveal any crystallographic phase separation and indicate that Mn occupies Ga-substitutional sites in the Mn concentration range up to 1%. The magnetic properties as a function of temperature, magnetic field and its orientation with respect to the c-axis of the wurtzite structure can be quantitatively described by the paramagnetic theory of an ensemble of non-interacting Mn$^{3+}$ ions in the relevant crystal field, a conclusion consistent with the x-ray absorption near edge structure analysis. A negligible contribution of Mn in the 2+ charge state points to a low concentration of residual donors in the studied films. Studies on modulation doped p-type (Ga,Mn)N/(Ga,Al)N:Mg heterostructures do not reproduce the high temperature robust ferromagnetism reported recently for this system.Comment: 15 pages, 14 figure

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects