DroplIT, an improved image analysis method for droplet identification in high-throughput crystallization trials

The application of robotics to protein crystallization trials has resulted in the production of millions of images. Manual inspection of these images to find crystals and other interesting outcomes is a major rate-limiting step. As a result there has been intense activity in developing automated algorithms to analyse these images. The very first step for most systems that have been described in the literature is to delineate each droplet. Here, a novel approach that reaches over 97% success rate and subsecond processing times is presented. This will form the seed of a new high-throughput system to scrutinize massive crystallization campaigns automatically. © 2010 International Union of Crystallography Printed in Singapore-all rights reserved.

Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5pl5. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Biophysical Characterization of Oxidized Phospholipids : Implications for Altered Membrane Structure and Function

The present study aims to elucidate the modifications in the structure and functionality of the phospholipid matrix of biological membranes brought about by free radical-mediated oxidative damage of its molecular constituents. To this end, the surface properties of two oxidatively modified phospholipids bearing an aldehyde or carboxyl function at the end of truncated sn-2 acyl chain were studied using a Langmuir balance. The results obtained reveal both oxidized species to have a significant impact on the structural dynamics of phospholipid monolayers, as illustrated by the progressive changes in force-area isotherms with increasing mole fraction of the oxidized lipid component. Moreover, surface potential measurements revealed considerable modifications in the electric properties of oxidized phospholipid containing monolayers during film compression, suggesting a packing state-controlled reorientation of the intramolecular electric dipoles of the lipid headgroups and acyl chains. Based on the above findings, a model describing the conformational state of oxidized phospholipid molecules in biological membranes is proposed, involving the protrusion of the acyl chains bearing the polar functional groups out from the hydrocarbon phase to the surrounding aqueous medium. Oxidative modifications alter profoundly the physicochemical properties of unsaturated phospholipids and are therefore readily anticipated to have important implications for their interactions with membrane-associating molecules. Along these lines, the carboxyl group bearing lipid was observed to bind avidly the peripheral membrane protein cytochrome c. The binding was reversed following increase in ionic strength or addition of polyanionic ATP, thus suggesting it to be driven by electrostatic interactions between cationic residues of the protein and the deprotonated lipid carboxyl exposed to the aqueous phase. The presence of aldehyde function bearing oxidized phospholipid was observed to enhance the intercalation of four antimicrobial peptides into phospholipid monolayers and liposomal bilayers. Partitioning of the peptides to monolayers was markedly attenuated by the aldehyde scavenger methoxyamine, revealing it to be mediated by the carbonyl moiety possibly through efficient hydrogen bonding or, alternatively, formation of covalent adduct in form of a Schiff base between the lipid aldehydes and primary amine groups of the peptide molecules. Lastly, both oxidized phospholipid species were observed to bind with high affinity three small membrane-partitioning therapeutic agents, viz. chlorpromazine, haloperidol, and doxorubicin. In conclusion, the results of studies conducted using biomimetic model systems support the notion that oxidative damage influences the molecular architecture as well as the bulk physicochemical properties of phospholipid membranes. Further, common polar functional groups carried by phospholipids subjected to oxidation were observed to act as molecular binding sites at the lipid-water interface. It is thus plausible that oxidized phospholipid species may elicit cellular level effects by modulating integration of various membrane-embedded and surface-associated proteins and peptides, whose conformational state, oligomerization, and functionality is known to be controlled by highly specific lipid-protein interactions and proper physical state of the membrane environment.

Chaperoning mitochondrial permeability transition: regulation of transition pore complex by a J-protein, DnaJC15

Mitochondria have a central role in the intrinsic pathway of apoptosis and involve activation of several transmembrane channels leading to release of death factors. Reduced expression of a mitochondrial J-protein DnaJC15 was associated with the development of chemoresistance in ovarian cancer cells. DnaJC15 was found to be a part of mitochondrial protein-transport machinery, though its connection with cell death mechanisms is still unclear. In the present study, we have provided evidence towards a novel function of DnaJC15 in regulation of mitochondrial permeability transition pore (MPTP) complex in normal and cancer cells. Overexpression of DnaJC15 resulted in MPTP opening and induction of apoptosis, whereas reduced amount of protein suppressed MPTP activation, upon cisplatin treatment. DnaJC15 was found to exert its proapoptotic function through the essential component of MPTP, cyclophilin D (CypD). Our results reveal a specific role of DnaJC15 in recruitment and coupling of CypD with mitochondrial permeability transition. In summary, our analysis provides first-time insights on the functional connection between mitochondrial inner membrane protein translocation machinery-associated J-protein DnaJC15 and regulation of cell death pathways.

Sequence and conformational preferences at termini of alpha-helices in membrane proteins: Role of the helix environment

-helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These -helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze -helices in a high-resolution dataset of integral -helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420-3436. (c) 2014 Wiley Periodicals, Inc.

Mycobacterium tuberculosis Cell Division Protein, FtsE, is an ATPase in Dimeric Form

FtsE is one of the earliest cell division proteins that assembles along with FtsX at the mid-cell site during cell division in Escherichia coli. Both these proteins are highly conserved across diverse bacterial genera and are predicted to constitute an ABC transporter type complex, in which FtsE is predicted to bind ATP and hydrolyse it, and FtsX is predicted to be an integral membrane protein. We had earlier reported that the MtFtsE of the human pathogen, Mycobacterium tuberculosis, binds ATP and interacts with MtFtsX on the cell membrane of M. tuberculosis and E. coli. In this study, we demonstrate that MtFtsE is an ATPase, the active form of which is a dimer, wherein the participating monomers are held together by non-covalent interactions, with the Cys84 of each monomer present at the dimer interface. Under oxidising environment, the dimer gets stabilised by the formation of Cys84-Cys84 disulphide bond. While the recombinant MtFtsE forms a dimer on the membrane of E. coli, the native MtFtsE seems to be in a different conformation in the M. tuberculosis membrane. Although disulphide bridges were not observed on the cytoplasmic side (reducing environment) of the membrane, the two participating monomers could be isolated as dimers held together by non-covalent interactions. Taken together, these findings show that MtFtsE is an ATPase in the non-covalent dimer form, with the Cys84 of each monomer present in the reduced form at the dimer interface, without participating in the dimerisation or the catalytic activity of the protein.

Residue proximity information and protein model discrimination using saturation-suppressor mutagenesis

Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proximal suppressors were accurately discriminated from distal suppressors based on their phenotypes when present as single mutants. Experimentally identified putative proximal pairs formed spatial constraints to recover >98% of native-like models of CcdB from a decoy dataset. Suppressor methodology was also applied to the integral membrane protein, diacylglycerol kinase A where the structures determined by X-ray crystallography and NMR were significantly different. Suppressor as well as sequence co-variation data clearly point to the Xray structure being the functional one adopted in vivo. The methodology is applicable to any macromolecular system for which a convenient phenotypic assay exists.

A study of protein targeting reveals insights into mitigating protein aggregation

A unique chloroplast Signal Recognition Particle (SRP) in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll-a/b binding (LHC) proteins. Our study of the thermodynamics and kinetics of the GTPases of the system demonstrates that GTPase complex assembly and activation are highly coupled in the chloroplast GTPases, suggesting they may forego the GTPase activation step as a key regulatory point. This reflects adaptations of the chloroplast SRP to the delivery of their unique substrate protein. Devotion to one highly hydrophobic family of proteins also may have allowed the chloroplast SRP system to evolve an efficient chaperone in the cpSRP43 subunit. To understand the mechanism of disaggregation, we showed that LHC proteins form micellar, disc-shaped aggregates that present a recognition motif (L18) on the aggregate surface. Further molecular genetic and structure-activity analyses reveal that the action of cpSRP43 can be dissected into two steps: (i) initial recognition of L18 on the aggregate surface; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. We also tested the adaptability of cpSRP43 for alternative substrates, specifically in attempts to improve membrane protein expression and inhibition of amyloid beta fibrillization. These preliminary results attest to cpSRP43’s potential as a molecular chaperone and provides the impetus for further engineering endeavors to address problems that stem from protein aggregation.

The Get3 ATPase drives unidirectional targeting of tail-anchored membrane proteins

Efficient and accurate localization of membrane proteins is essential to all cells and requires a complex cascade of interactions between protein machineries. This is exemplified in the recently discovered Guided Entry of Tail-anchored protein pathway, in which the central targeting factor Get3 must sequentially interact with three distinct binding partners (Get4, Get1 and Get2) to ensure the targeted delivery of Tail-anchored proteins to the endoplasmic reticulum membrane. To understand the molecular and energetic principles that provide the vectorial driving force of these interactions, we used a quantitative fluorescence approach combined with mechanistic enzymology to monitor the effector interactions of Get3 at each stage of Tail-anchored protein targeting. We show that nucleotide and membrane protein substrate generate a gradient of interaction energies that drive the cyclic and ordered transit of Get3 from Get4 to Get2 and lastly to Get1. These data also define how the Get3/Tail-anchored complex is captured, handed over, and disassembled by the Get1/2 receptor at the membrane, and reveal a novel role for Get4/5 in recycling Get3 from the endoplasmic reticulum membrane at the end of the targeting reaction. These results provide general insights into how complex cascades of protein interactions are coordinated and coupled to energy inputs in biological systems.

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Flavobacterium columnare

Outer membrane proteins (OMPs) of bacteria are key molecules interacting with the host environment. Flavobacterium columnare, a pathogen-causing columnaris disease of fish worldwide, was studied in order to understand the composition of its OMPs. The sarcosine-insoluble membrane fraction of the OMPs was analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with reverse-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC MS/MS). Thirty-six proteins were identified, including proteins involved in cell wall/membrane biogenesis, specific transport of various nutrients and in essential metabolism. The present study is the first report on the OMPs of F. columnare, and may serve as the basis for understanding the pathogenesis of the bacterium.

De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex.

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

Functional characterization and membrane topology of Escherichia coli WecA, a sugar-phosphate transferase initiating the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide

WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K(m) and V(max) values for UDP-GlcNAc, Mg(2+), and Mn(2+) suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg(2+), possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli

The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

WecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate N-acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in Escherichia coli and other enteric bacteria. WecA functions as an UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279-282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH279-282 and the Arg265 are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the E. coli wecA mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H279) of the HIHH motif as well as the Arg265 residue. The functional importance of these residues is underscored by the high level of conservation of H279 and Arg265 among bacterial WecA homologues that utilize several different UDP-N-acetylhexosamine substrates.

Outer membrane proteins of Bacteroides fragilis grown in vivo

The outer membrane protein (OMP) profiles of four different strains of Bacteroides fragilis, as determined by Coomassie blue stained polyacrylamide gels, were compared after growth in broth culture and in the mouse peritoneal cavity. There was no induction of the expression of large quantities of novel OMP after growth in vivo. Mouse immunoglobulin G and albumin were associated with the bacterial OMP, but could be removed by washing.