Crystal structures of fusion proteins with large-affinity tags

The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.

Comparison of three commercial sparse-matrix crystallization screens

Sparse-matrix sampling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 diverse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.

Structural studies of copper(II)-amine terminated dendrimer complexes by EXAFS

The three-dimensional branched nature of dendritic macromolecules provides many potential sites per molecule for the complexation of metal ions. Therefore, dendrimers may act as hosts for metals with coordination potentially occurring at the periphery, the interior, or both. To understand further the complexation of dendrimers with metal ions EXAFS experiments were carried out. In this work, the interaction of amine-terminated polyamido(amine), PAMAM, dendrimer with copper(II) ions determined by EXAFS is reported. It was found that a model consisting of the copper(II) ion forming five- and six-membered rings by chelating with the primary amine, amide, and tertiary amine nitrogen donors of the PAMAM dendrimer could describe the experimental EXAFS data well. Corroborative evidence for binding to amide nitrogen donors comes from the broadening of NMR resonances of a copper(Il)-PAMAM mixture revealing the presence of paramagnetic copper(II) ions at these sites. The significance of the results presented in this paper is that copper(II) ions form complexes within the dendrimer structure and not just at the periphery. The current study may have implications for the use of PAMAM dendrimers as effective ligands in sensing systems.

Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.

Protein contact prediction using patterns of correlation

We describe a new method for using neural networks to predict residue contact pairs in a protein. The main inputs to the neural network are a set of 25 measures of correlated mutation between all pairs of residues in two windows of size 5 centered on the residues of interest. While the individual pair-wise correlations are a relatively weak predictor of contact, by training the network on windows of correlation the accuracy of prediction is significantly improved. The neural network is trained on a set of 100 proteins and then tested on a disjoint set of 1033 proteins of known structure. An average predictive accuracy of 21.7% is obtained taking the best L/2 predictions for each protein, where L is the sequence length. Taking the best L/10 predictions gives an average accuracy of 30.7%. The predictor is also tested on a set of 59 proteins from the CASP5 experiment. The accuracy is found to be relatively consistent across different sequence lengths, but to vary widely according to the secondary structure. Predictive accuracy is also found to improve by using multiple sequence alignments containing many sequences to calculate the correlations. (C) 2004 Wiley-Liss, Inc.

Crystallization and preliminary X-ray diffraction analysis of importin-alpha acomplexed with NLS peptidomimetics

Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). Tile study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-a was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 angstrom resolution. Preliminary electron density calculations show that the ligands are present in the crystals. (c) 2005 Elsevier B.V All rights reserved.

Alkali hydroxide-induced gelation of pectin

The mechanism of pectin gelation depends on the degree of methoxylation. High methoxyl pectin gels due to hydrophobic interactions and hydrogen bonding between pectin molecules. Low methoxyl pectin forms gels in the presence of di- and polyvalent cations which cross link and neutralise the negative charges of the pectin molecule. Monovalent cations normally do not lead to gel formation with high methoxyl pectin solutions free of divalent cations, especially Ca. The present study found that alkali (NaOH or KOH) added to high methoxyl pectin leads to gel formation in a concentration-depended manner. It was also found that monovalent cations (Na and K) induce gelation of low methoxyl pectin and the time required for gel formation (setting time) depends on the cation concentration. The results indicate that a combined char-e neutralisation and ionic strength effect is responsible for the monovalent cation-induced gelation of pectin. (C) 2003 Elsevier Ltd. All rights reserved.

A study of the effects of postcure treatments on polyester-melamine coating matrices

The effect of postcure high energy (gamma), ultraviolet (UV) and thermal treatment on the properties of polyester-melamine clearcoats of a range of compositions has been investigated. Two initial cure conditions were used, of which one was '' optimally '' cured and the other undercured. It was found that postcure treatments, particularly gamma and UV, led to coatings of similar mechanical and thermal properties irrespective of initial cure, although the change in properties on postcure treatment was greater for the under-cured samples. The results were interpreted in terms of the effect of the treatments on the structure of the crosslinked matrices. The study suggests the possibility of the development of a dual-cure process for polyester-melamines, whereby cure optimization and property improvement can be achieved. This could also be used to '' correct '' for small variations in thermal cure levels brought about by adventitious online fluctuations in cure oven conditions.

Controlled release of heparin from poly(epsilon-caprolactone) electrospun fibers

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly(epsilon-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8% w/v PCL in 7:3 dichloromethane: methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury. (C) 2005 Elsevier Ltd. All rights reserved.

Crystallization of the C-terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase

The mammalian long-chain acyl-CoA thioesterase, the enzyme that catalyses the hydrolysis of acyl-CoAs to free fatty acids, contains two fused 4HBT (4-hydroxybenzoyl-CoA thioesterase) motifs. The C-terminal domain of the mouse long-chain acyl-CoA thioesterase (Acot7) has been expressed in bacteria and crystallized. The crystals were obtained by vapour diffusion using PEG 2000 MME as precipitant at pH 7.0 and 290 K. The crystals have the symmetry of space group R32 ( unit-cell parameters a = b = 136.83, c = 99.82 angstrom, gamma = 120 degrees). Two molecules are expected in the asymmetric unit. The crystals diffract to 2.4 angstrom resolution using the laboratory X-ray source and are suitable for crystal structure determination.

Efficient -tensor determination and NH assignment of paramagnetic proteins

Anisotropic magnetic susceptibility tensors chi of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the chi-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D N-15-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable chi-tensor anisotropy parameters from 2D spectra of uniformly N-15-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit epsilon from E. coli DNA polymerase III in complex with the subunit theta and La3+ in its diamagnetic and Dy3+, Tb3+, and Er3+ in its paramagnetic form.

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 angstrom r.m.s. deviation of C alpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.

Site-specific labelling of proteins with a rigid lanthanide-binding tag

This paper describes a generic method for the site-specific attachment of lathanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Echerichia coli arginine repressor. Complexes with Y3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+ and Yb3+ ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisostropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Introducing amine functionalities on a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) surface: Comparing the use of ammonia plasma treatment and ethylenediamine aminolysis

Amine functionalities were introduced onto the surface of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films by applying radio frequency ammonia plasma treatment and wet ethylenediamine treatment. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS) for chemical composition and Raman microspectroscopy for the spatial distribution of the chemical moieties. The relative amount of amine functionalities introduced onto the PHBV surface was determined by exposing the treated films to the vapor of trifluoromethylbenzaldehyde (TFBA) prior to XPS analysis. The highest amount of amino groups on the PHBV surface could be introduced by use of ammonia plasma at short treatment times of 5 and 10 s, but no effect of plasma power within the range of 2.5-20 W was observed. Ethylenediamine treatment yielded fewer surface amino groups, and in addition an increase in crystallinity as well as degradation of PHBV was evident from Fourier transform infrared spectroscopy. Raman maps showed that the coverage of amino groups on the PHBV surfaces was patchy with large areas having no amine functionalities.