Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ~55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.
Copyright © 2010 The Protein Society.lørdag 12. mars 2011
Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.
Modeling of the Toll-like receptor 3 and a putative Toll-like receptor 3 antagonist encoded by the African swine fever virus.
African swine fever virus (ASFV) is a large double-stranded DNA virus responsible for a lethal pig disease, to which no vaccine has ever been obtained. Its genome encodes a number of proteins involved in virus survival and transmission in its hosts, in particular proteins that inhibit signaling pathways in infected macrophages and, thus, interfere with the host's innate immune response. A recently identified novel ASFV viral protein (pI329L) was found to inhibit the Toll-like receptor 3 (TLR3) signaling pathway, TLR3 being a crucial "danger detector." pI329L has been predicted to be a transmembrane protein containing extracellular putative leucine-rich repeats similar to TLR3, suggesting that pI329L might act as a TLR3 decoy. To explore this idea, we used comparative modeling and other structure prediction protocols to propose (a) a model for the TLR3-Toll-interleukin-1 receptor homodimer and (b) a structural fold for pI329L, detailed at atomistic level for its cytoplasmic domain. As this later domain shares only remote sequence relationships with the available TLR3 templates, a more complex modeling strategy was employed that combines the iterative implementation of (multi)threading/assembly/refinement (I-TASSER) structural prediction with expertise-guided posterior refinement. The final pI329L model presents a plausible fold, good structural quality, is consistent with the available experimental data, and it corroborates our hypothesis of pI329L being a TLR3 antagonist.
Copyright © 2010 The Protein Society.Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21.
Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.
Copyright © 2010 The Protein Society.fredag 11. mars 2011
Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 A resolution.
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2 arachidonoyl glycerol. A model is proposed in which monoglyceride lipase undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Copyright © 2011 The Protein Society.PMID: 21308848 [PubMed - as supplied by publisher]Calorimetric study of a series of designed repeat proteins: Modular structure and modular folding.
Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.
Copyright © 2010 The Protein Society.Solution structure and fluctuation of the Mg(2+) -bound form of Calmodulin C-terminal domain.
Calmodulin (CaM) is a Ca(2+) -binding protein that functions as a ubiquitous Ca(2+) -signaling molecule, through conformational changes from the "closed" apo conformation to the "open" Ca(2+) -bound conformation. Mg(2+) also binds to CaM and stabilizes its folded structure, but the NMR signals are broadened by slow conformational fluctuations. Using the E104D/E140D mutant, designed to decrease the signal broadening in the presence of Mg(2+) with minimal perturbations of the overall structure, the solution structure of the Mg(2+) -bound form of the CaM C-terminal domain was determined by multidimensional NMR spectroscopy. The Mg(2+) -induced conformational change mainly occurred in EF hand IV, while EF-hand III retained the apo structure. The helix G and helix H sides of the binding sequence undergo conformational changes needed for the Mg(2+) coordination, and thus the helices tilt slightly. The aromatic rings on helix H move to form a new cluster of aromatic rings in the hydrophobic core. Although helix G tilts slightly to the open orientation, the closed conformation is maintained. The fact that the Mg(2+) -induced conformational changes in EF-hand IV and the hydrophobic core are also seen upon Ca(2+) binding suggests that the Ca(2+) -induced conformational changes can be divided into two categories, those specific to Ca(2+) and those common to Ca(2+) and Mg(2+) .
Copyright © 2011 The Protein Society.torsdag 10. mars 2011
Structural determinants of ligand imprinting: A molecular dynamics simulation study of subtilisin in aqueous and apolar solvents.
The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.
Copyright © 2010 The Protein Society.Residual interactions in unfolded bile acid-binding protein by (19) F NMR.
The folding initiation mechanism of human bile acid-binding protein (BABP) has been examined by (19) F NMR. Equilibrium unfolding studies of BABP labeled with fluorine at all eight of its phenylalanine residues showed that at least two sites experience changes in solvent exposure at high denaturant concentrations. Peak assignments were made by site-specific 4FPhe incorporation. The resonances for proteins specifically labeled at Phe17, Phe47, and Phe63 showed changes in chemical shift at denaturant concentrations at which the remaining five phenylalanine residues appear to be fully solvent-exposed. Phe17 is a helical residue that was not expected to participate in a folding initiation site. Phe47 and Phe63 form part of a hydrophobic core region that may be conserved as a site for folding initiation in the intracellular lipid-binding protein family.
Copyright © 2010 The Protein Society.A secretory system for bacterial production of high-profile protein targets.
Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters a nd selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.
Copyright © 2011 The Protein Society.onsdag 9. mars 2011
The dynameomics rotamer library: Amino acid side chain conformations and dynamics from comprehensive molecular dynamics simulations in water.
We have recently completed systematic molecular dynamics simulations of 807 different proteins representing 95% of the known autonomous protein folds in an effort we refer to as Dynameomics. Here we focus on the analysis of side chain conformations and dynamics to create a dynamic rotamer library. Overall this library is derived from 31,000 occurrences of each of 86,217 different residues, or 2.7 × 10(9) rotamers. This dynamic library has 74% overlap of rotamer distributions with rotamer libraries derived from static high-resolution crystal structures. Seventy-five percent of the residues had an assignable primary conformation, and 68% of the residues had at least one significant alternate conformation. The average correlation time for switching between rotamers ranged from 22 ps for Met to over 8 ns for Cys; this time decreased 20-fold on the surface of the protein and modestly for dihedral angles further from the main chain. Side chain S(2) axis order parameters were calculated and they correlated well with those derived from NMR relaxation experiments (R = 0.9). Relationships relating the S(2) axis order parameters to rotamer occupancy were derived. Overall the Dynameomics rotamer library offers a comprehensive depiction of side chain rotamer preferences and dynamics in solution, and more realistic distributions for dynamic proteins in solution at ambient temperature than libraries derived from crystal structures, in particular charged surface residues are better represented. Details of the rotamer library are presented here and the library itself can be downloaded at http://www.dynameomics.org.
Copyright © 2010 The Protein Society.tirsdag 8. mars 2011
Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor and spin labels.
Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid ß-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (aII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for P. denitrificans and human ETF. To characterize the orientations of the aII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8±0.15 nm for the label in the FAD-containing aII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm) and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in aI (A43C) were between 4.0 and 4.5±0.35 nm and for ßIII (A111C) the distance was 4.3±0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the aII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open).
Copyright © 2011 The Protein Society.Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide.
The human major histocompatibility complex class I antigen HLA-B*2705 binds several sequence-related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross-reactivity of cytotoxic T cells (CTL) against these HLA-B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging-in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross-reactivity of these CTL with a peptide derived from voltage-dependent calcium channel a1 subunit (pCAC, SRRWRRWNR), in which the C-terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA-B*2705:pCAC complex by X-ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence-related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F-pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA-B*2705 heavy chain near the N-terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg-Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR-directed, HLA-B*2705-restricted CTL to cross-react with HLA-B*2705:pCAC complexes.
Copyright © 2010 The Protein Society.A secretory system for bacterial production of high-profile protein targets.
Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters a nd selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.
Copyright © 2011 The Protein Society.mandag 7. mars 2011
NMR determination of pK(a) values in alpha-synuclein.
The intrinsically unfolded protein a-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For a-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the a-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed a-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the a-synuclein sequence may help nucleate the folding of the protein into an a-helical structure and confer protection from misfolding.
Copyright © 2010 The Protein Society.2-Methyl-2,4-pentanediol induces spontaneous assembly of staphylococcal alpha-hemolysin into heptameric pore structure.
Staphylococcal a-hemolysin is expressed as a water-soluble monomeric protein and assembles on membranes to form a heptameric pore structure. The heptameric pore structure of a-hemolysin can be prepared from monomer in vitro only in the presence of deoxycholate detergent micelles, artificially constructed phospholipid bilayers, or erythrocytes. Here, we succeeded in preparing crystals of the heptameric form of a-hemolysin without any detergent but with 2-methyl-2,4-pentanediol (MPD), and determined its structure. The structure of the heptameric pore was similar to that reported previously. In the structure, two molecules of MPD were bound around Trp179, around which phospholipid head groups were bound in the heptameric pore structure reported previously. Size exclusion chromatography showed that a-hemolysin did not assemble spontaneously even when stored for 1 year. SDS-PAGE analysis revealed that, among the compounds in the crystallizing buffer, MPD could induce heptamer formation. The concentration of MPD that most efficiently induced oligomerization was between 10 and 30%. Based on these observations, we propose MPD as a reagent that can facilitate heptameric pore formation of a-hemolysin without membrane binding.
Copyright © 2011 The Protein Society.søndag 6. mars 2011
Conserved core of amyloid fibrils of wild type and A30P mutant alpha-synuclein.
The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein a-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected high-resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of ß-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates.
Copyright © 2010 The Protein Society.Counting peptide-water hydrogen bonds in unfolded proteins.
It is often assumed that the peptide backbone forms a substantial number of additional hydrogen bonds when a protein unfolds. We challenge that assumption in this article. Early surveys of hydrogen bonding in proteins of known structure typically found that most, but not all, backbone polar groups are satisfied, either by intramolecular partners or by water. When the protein is folded, these groups form approximately two hydrogen bonds per peptide unit, one donor or acceptor for each carbonyl oxygen or amide hydrogen, respectively. But when unfolded, the backbone chain is often believed to form three hydrogen bonds per peptide unit, one partner for each oxygen lone pair or amide hydrogen. This assumption is based on the properties of small model compounds, like N-methylacetamide, or simply accepted as self-evident fact. If valid, a chain of N residues would have approximately 2N backbone hydrogen bonds when folded but 3N backbone hydrogen bonds when unfolded, a sufficient difference to overshadow any uncertainties involved in calculating these per-residue averages. Here, we use exhaustive conformational sampling to monitor the number of H-bonds in a statistically adequate population of blocked polyalanyl-six-mers as the solvent quality ranges from good to poor. Solvent quality is represented by a scalar parameter used to Boltzmann-weight the population energy. Recent experimental studies show that a repeating (Gly-Ser) polypeptide undergoes a denaturant-induced expansion accompanied by breaking intramolecular peptide H-bonds. Results from our simulations augment this experimental finding by showing that the number of H-bonds is approximately conserved during such expansion?compaction transitions.
Copyright © 2010 The Protein Society.A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data.
The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X-ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low-resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two-dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low-resolution phase information that can be refined further by the conventional methods of protein crystallography.
Copyright © 2011 The Protein Society.lørdag 5. mars 2011
Crystal structure of the Nogo-receptor-2.
The inhibition of axon regeneration upon mechanical injury is dependent on interactions between Nogo receptors (NgRs) and their myelin-derived ligands. NgRs are composed of a leucine-rich repeat (LRR) region, thought to be structurally similar among the different isoforms of the receptor, and a divergent "stalk" region. It has been shown by others that the LRR and stalk regions of NgR1 and NgR2 have distinct roles in conferring binding affinity to the myelin associated glycoprotein (MAG) in vivo. Here we show that purified recombinant full length NgR1 and NgR2 maintain significantly higher binding affinity for purified MAG as compared to the isolated LRR region of either NgR1 or NgR2. We also present the crystal structure of the LRR and part of the stalk regions of NgR2 and compare it to the previously reported NgR1 structure with respect to the distinct signaling properties of the two receptor isoforms.
Copyright © 2011 The Protein Society.Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes.
Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ~40, 30, and 0.09 µM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1.
Copyright © 2010 The Protein Society.fredag 4. mars 2011
Conserved core of amyloid fibrils of wild type and A30P mutant alpha-synuclein.
The major component of neural inclusions that are the pathological hallmark of Parkinson's disease are amyloid fibrils of the protein a-synuclein (aS). Here we investigated if the disease-related mutation A30P not only modulates the kinetics of aS aggregation, but also alters the structure of amyloid fibrils. To this end we optimized the method of quenched hydrogen/deuterium exchange coupled to NMR spectroscopy and performed two-dimensional proton-detected high-resolution magic angle spinning experiments. The combined data indicate that the A30P mutation does not cause changes in the number, location and overall arrangement of ß-strands in amyloid fibrils of aS. At the same time, several residues within the fibrillar core retain nano-second dynamics. We conclude that the increased pathogenicity related to the familial A30P mutation is unlikely to be caused by a mutation-induced change in the conformation of aS aggregates.
Copyright © 2010 The Protein Society.Counting peptide-water hydrogen bonds in unfolded proteins.
It is often assumed that the peptide backbone forms a substantial number of additional hydrogen bonds when a protein unfolds. We challenge that assumption in this article. Early surveys of hydrogen bonding in proteins of known structure typically found that most, but not all, backbone polar groups are satisfied, either by intramolecular partners or by water. When the protein is folded, these groups form approximately two hydrogen bonds per peptide unit, one donor or acceptor for each carbonyl oxygen or amide hydrogen, respectively. But when unfolded, the backbone chain is often believed to form three hydrogen bonds per peptide unit, one partner for each oxygen lone pair or amide hydrogen. This assumption is based on the properties of small model compounds, like N-methylacetamide, or simply accepted as self-evident fact. If valid, a chain of N residues would have approximately 2N backbone hydrogen bonds when folded but 3N backbone hydrogen bonds when unfolded, a sufficient difference to overshadow any uncertainties involved in calculating these per-residue averages. Here, we use exhaustive conformational sampling to monitor the number of H-bonds in a statistically adequate population of blocked polyalanyl-six-mers as the solvent quality ranges from good to poor. Solvent quality is represented by a scalar parameter used to Boltzmann-weight the population energy. Recent experimental studies show that a repeating (Gly-Ser) polypeptide undergoes a denaturant-induced expansion accompanied by breaking intramolecular peptide H-bonds. Results from our simulations augment this experimental finding by showing that the number of H-bonds is approximately conserved during such expansion?compaction transitions.
Copyright © 2010 The Protein Society.torsdag 3. mars 2011
Structural characterization reveals that a PilZ domain protein undergoes substantial conformational change upon binding to cyclic dimeric guanosine monophosphate.
PA4608 is a single PilZ domain protein from Pseudomonas aeruginosa that binds to cyclic dimeric guanosine monophosphate (c-di-GMP). Although the monomeric structure of unbound PA4608 has been studied in detail, the molecular details of c-di-GMP binding to this protein are still uncharacterized. Hence, we determined the solution structure of c-di-GMP bound PA4608. We found that PA4608 undergoes conformational changes to expose the c-di-GMP binding site by ejection of the C-terminal 3(10) helix. A dislocation of the C-terminal tail in the presence of c-di-GMP implies that this region acts as a lid that alternately covers and exposes the hydrophobic surface of the binding site. In addition, mutagenesis and NOE data for PA4608 revealed that conserved residues are in contact with the c-di-GMP molecule. The unique structural characteristics of PA4608, including its monomeric state and its ligand binding characteristics, yield insight into its function as a c-di-GMP receptor.
Copyright © 2010 The Protein Society.Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures.
Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10) - and a-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.
Copyright © 2010 The Protein Society.onsdag 2. mars 2011
Crystal structure of the functional region of Uro-adherence factor A from Staphylococcus saprophyticus reveals participation of the B domain in ligand binding.
Staphylococci use cell wall-anchored proteins as adhesins to attach to host tissues. Staphylococcus saprophyticus, a uropathogenic species, has a unique cell wall-anchored protein, uro-adherence factor A (UafA), which shows erythrocyte binding activity. To investigate the mechanism of adhesion by UafA, we determined the crystal structure of the functional region of UafA at 1.5 Å resolution. The structure was composed of three domains, designated as the N2, N3, and B domains, arranged in a triangular relative configuration. Hemagglutination inhibition assay with domain-truncated mutants indicated that both N and B domains were necessary for erythrocyte binding. Based on these results, a novel manner of ligand binding in which the B domain acts as a functional domain was proposed as the adhesion mechanism of S. saprophyticus.
Copyright © 2010 The Protein Society.Protein disulfide isomerase (PDI) isomerizes non-native disulfide bonds in human proinsulin independent of its peptide binding activity.
Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.
Copyright © 2011 The Protein Society.OCAM: A new tool for studying the oligomeric diversity of MscL channels.
We have developed a new technique to study the oligomeric state of proteins in solution. OCAM or Oligomer Characterization by Addition of Mass counts protein subunits by selectively shaving a protein mass tag added to a protein subunit via a short peptide linker. Cleavage of each mass tag reduces the total mass of the protein complex by a fixed amount. By performing limited proteolysis and separating the reaction products by size on a blue native PAGE gel, a ladder of reaction products corresponding to the number of subunits can be resolved. The pattern of bands may be used to distinguish the presence of a single homo-oligomer from a mixture of oligomeric states. We have applied OCAM to study the mechanosensitive channel of large conductance (MscL) and find that these proteins can exist in multiple oligomeric states ranging from tetramers up to possible hexamers. Our results demonstrate the existence of oligomeric forms of MscL not yet observed by X-ray crystallography or other techniques and that in some cases a single type of MscL subunit can assemble as a mixture of oligomeric states.
Copyright © 2010 The Protein Society.tirsdag 1. mars 2011
A new approach for structure analysis of two-dimensional membrane protein crystals using X-ray powder diffraction data.
The application of powder diffraction methods to problems in structural biology is generally regarded as intractable because of the large number of unresolved, overlapping X-ray reflections. Here, we use information about unit cell lattice parameters, space group transformations, and chemical composition as a priori information in a bootstrap process that resolves the ambiguities associated with overlapping reflections. The measured ratios of reflections that can be resolved experimentally are used to refine the position, the shape, and the orientation of low-resolution molecular structures within the unit cell, in leading to the resolution of the overlapping reflections. The molecular model is then made progressively more sophisticated as additional diffraction information is included in the analysis. We apply our method to the recovery of the structure of the bacteriorhodopsin molecule (bR) to a resolution of 7 Å using experimental data obtained from two-dimensional purple membrane crystals. The approach can be used to determine the structure factors directly or to provide reliable low-resolution phase information that can be refined further by the conventional methods of protein crystallography.
Copyright © 2011 The Protein Society.Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition.
Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ~50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ~30 min with copper alone. Nickel had the largest effect, reducing the half-life to ~5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.
Copyright © 2010 The Protein Society.mandag 28. februar 2011
Binding of small molecules to cavity forming mutants of a de novo designed protein.
A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis, will require proteins that recognize and bind to small molecules. Here we show that stably folded a-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this 4-helix bundle. The parental protein and the Phe?Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference (STD) NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo (PFGSE) NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.
Copyright © 2011 The Protein Society.Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 A resolution.
A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2 arachidonoyl glycerol. A model is proposed in which monoglyceride lipase undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.
Copyright © 2011 The Protein Society.PMID: 21308848 [PubMed - as supplied by publisher]søndag 27. februar 2011
2-Methyl-2,4-pentanediol induces spontaneous assembly of staphylococcal alpha-hemolysin into heptameric pore structure.
Staphylococcal a-hemolysin is expressed as a water-soluble monomeric protein and assembles on membranes to form a heptameric pore structure. The heptameric pore structure of a-hemolysin can be prepared from monomer in vitro only in the presence of deoxycholate detergent micelles, artificially constructed phospholipid bilayers, or erythrocytes. Here, we succeeded in preparing crystals of the heptameric form of a-hemolysin without any detergent but with 2-methyl-2,4-pentanediol (MPD), and determined its structure. The structure of the heptameric pore was similar to that reported previously. In the structure, two molecules of MPD were bound around Trp179, around which phospholipid head groups were bound in the heptameric pore structure reported previously. Size exclusion chromatography showed that a-hemolysin did not assemble spontaneously even when stored for 1 year. SDS-PAGE analysis revealed that, among the compounds in the crystallizing buffer, MPD could induce heptamer formation. The concentration of MPD that most efficiently induced oligomerization was between 10 and 30%. Based on these observations, we propose MPD as a reagent that can facilitate heptameric pore formation of a-hemolysin without membrane binding.
Copyright © 2011 The Protein Society.Insights into the conformational flexibility of Bruton's tyrosine kinase from multiple ligand complex structures.
Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10) - and a-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors.
Copyright © 2010 The Protein Society.Structures of domains I and IV from YbbR are representative of a widely distributed protein family.
YbbR domains are widespread throughout Eubacteria and are expressed as monomeric units, linked in tandem repeats or cotranslated with other domains. Although the precise role of these domains remains undefined, the location of the multiple YbbR domain-encoding ybbR gene in the Bacillus subtilis glmM operon and its previous identification as a substrate for a surfactin-type phosphopantetheinyl transferase suggests a role in cell growth, division, and virulence. To further characterize the YbbR domains, structures of two of the four domains (I and IV) from the YbbR-like protein of Desulfitobacterium hafniense Y51 were solved by solution nuclear magnetic resonance and X-ray crystallography. The structures show the domains to have nearly identical topologies despite a low amino acid identity (23%). The topology is dominated by ß-strands, roughly following a "figure 8" pattern with some strands coiling around the domain perimeter and others crossing the center. A similar topology is found in the C-terminal domain of two stress-responsive bacterial ribosomal proteins, TL5 and L25. Based on these models, a structurally guided amino acid alignment identifies features of the YbbR domains that are not evident from naïve amino acid sequence alignments. A structurally conserved cis-proline (cis-Pro) residue was identified in both domains, though the local structure in the immediate vicinities surrounding this residue differed between the two models. The conservation and location of this cis-Pro, plus anchoring Val residues, suggest this motif may be significant to protein function.
Copyright © 2011 The Protein Society.lørdag 26. februar 2011
Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation.
Sparsely populated states of macromolecules, characterized by short lifetimes and high free-energies relative to the predominant ground state, often play a key role in many biological, chemical, and biophysical processes. In this review, we briefly summarize various new developments in NMR spectroscopy that permit these heretofore invisible, sparsely populated states to be detected, characterized, and in some instances visualized. Relaxation dispersion spectroscopy yields detailed kinetic information on processes involving species characterized by distinct chemical shifts with lifetimes in the ~50 µs-10 ms range and populations as low as 0.5%. In the fast exchange regime (time scale less than ~250-500 µs), the footprint of sparsely populated states can be observed on paramagnetic relaxation enhancement profiles measured on the resonances of the major species, thereby yielding structural information that is directly related to paramagnetic center-nuclei distances from which it is possible, under suitable circumstances, to compute a structure or ensemble of structures for the minor species. Finally, differential transverse relaxation measurements can be used to detect lifetime broadening effects that directly reflect the unidirectional rates for the conversion of NMR-visible into high-molecular weight NMR-invisible species. Examples of these various approaches are presented.
Copyright © 2010 The Protein Society.Calorimetric study of a series of designed repeat proteins: Modular structure and modular folding.
Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.
Copyright © 2010 The Protein Society.fredag 25. februar 2011
Binding of small molecules to cavity forming mutants of a de novo designed protein.
A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis, will require proteins that recognize and bind to small molecules. Here we show that stably folded a-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this 4-helix bundle. The parental protein and the Phe?Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference (STD) NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo (PFGSE) NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.
Copyright © 2011 The Protein Society.Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide.
The human major histocompatibility complex class I antigen HLA-B*2705 binds several sequence-related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross-reactivity of cytotoxic T cells (CTL) against these HLA-B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging-in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross-reactivity of these CTL with a peptide derived from voltage-dependent calcium channel a1 subunit (pCAC, SRRWRRWNR), in which the C-terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA-B*2705:pCAC complex by X-ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence-related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F-pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA-B*2705 heavy chain near the N-terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg-Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR-directed, HLA-B*2705-restricted CTL to cross-react with HLA-B*2705:pCAC complexes.
Copyright © 2010 The Protein Society.torsdag 24. februar 2011
Crystal structures of CmeR-bile acid complexes from Campylobacter jejuni.
The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acid-binding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.
Copyright © 2011 The Protein Society.Potenspiller
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The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS.
Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N(2) ? I(2) ? 2U) process involving a dimeric intermediate (I(2) ). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N(2) ? I(2) transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.
Copyright © 2010 The Protein Society.onsdag 23. februar 2011
Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition.
Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ~50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ~30 min with copper alone. Nickel had the largest effect, reducing the half-life to ~5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.
Copyright © 2010 The Protein Society.Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR-like protein family.
The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadC(pd) , is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadC(pd) (residues 188-512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl(6) (2-) derivative. CadC(pd) reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a $\tilde \beta$sheet in contact with three a-helices and a C-terminal subdomain formed by a ten-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in the heavy metal derivative of CadC(pd) used for MAD phasing a ReCl(6) (2-) ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadC(pd) and its variants form dimers in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.
Copyright © 2011 The Protein Society.Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.
Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ~55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.
Copyright © 2010 The Protein Society.torsdag 6. januar 2011
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