Simple Host-Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way

Time-Controlled Microfluidic Seeding in nL-Volume Droplets To Separate Nucleation and Growth Stages of Protein Crystallization

This paper describes a method of time-controlled seeding to separate the stages of nucleation and growth in protein crystallization using a microfluidic device

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 ×100 μm, belonged to space group P43212, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C380, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades

Crystal structures of B-DNA with incorporated 2′-deoxy-2′-fluoro-arabino-furanosyl thymines: Implications of conformational preorganization for duplex stability

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2′ position, such as in the 2′-deoxy-2′-fluororibofuranosyl analogue, will drive the conformational equilibrium towards the C3′-endo type (north). Conversely, the 2′-deoxy-2′-fluoroarabinofuranosyl modification with opposite stereochemistry at C2′ appears to have a preference for a C2′-endo type pucker (south). Incorporation of 2′-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2 and [d(CGCGAASTCGCG)]2 with incorporated 2′-deoxy-2′-fluoroarabinofuranosyl thymines S (south) at 1.55 Å resolution. In the crystal structures, all S residues adopt an O4′-endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2′-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2′-deoxy-2′-fluoroarabino thymidine

Crystal structures of the catalytic domain of human protein kinase associated with apoptosis and tumor suppression. Nature Structural & Molecular Biology

articles Programmed cell death (apoptosis) is key to normal development, and alterations of its control have been implicated in human disease, including the loss of tumor suppression linked to cancer progressio

Malignant Brain Tumor Repeats: A Three-Leaved Propeller Architecture with Ligand/Peptide Binding Pockets

We report on the X-ray structure of three 100-amino acid mbt repeats in h-l(3)mbt, a polycomb group protein involved in transcriptional repression, whose gene is located in a region of chromosome 20 associated with hematopoietic malignancies. Interdigitation between the extended arms and cores of the mbt repeats results in a three-leaved propeller-like architecture, containing a central cavity. We have identified one ligand binding pocket per mbt repeat, which accommodates either the morphilino ring of MES or the proline ring of the C-terminal peptide segment, within a cavity lined by aromatic amino acids. Strikingly, phenotypic alterations resulting from point mutations or deletions in the mbt repeats of the related Drosophila SCM protein are clustered in and around the ligand binding pocket