bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein
Authors: Broos Jaap , Gabellieri Edi , Biemans‐Oldehinkel Esther , Strambini Giovanni B. 
Published: August 2003

AbstractBiosynthetic incorporation of tryptophan (Trp) analogs such as 7‐azatryptophan, 5‐hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EIImtl). EIImtl overexpression was under control of the λPR promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the λPR promoter. Together with the presence of the repressor gene on the EIImtl plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EIImtl samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L‐Trp analogs.
The binding of β‐ and γ‐cyclodextrins to glycogen phosphorylase b: Kinetic and crystallographic studies
Authors: Pinotsis Nikos , Leonidas Demetres D. , Chrysina Evangelia D. , Oikonomakos Nikos G. , Mavridis Irene M. 
Published: August 2003

AbstractA number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, α‐, β‐, and γ‐cyclodextrins were identified as moderate mixed‐type competitive inhibitors of GPb (with respect to glycogen) with Ki values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with β‐ and γ‐cyclodextrins at 1.94 Å and 2.3 Å resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T‐state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that β‐ andγ‐cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of α‐cyclodextrin and octakis (2,3,6‐tri‐O‐methyl)‐γ‐cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.
Backbone and side–chain heteronuclear resonance assignments and hyperfine NMR shifts in horse cytochrome c
Authors: Liu Weixia , Rumbley Jon , Englander S. Walter , Wand A. Joshua 
Published: August 2003

AbstractThe [H26N, H33N] mutant of horse heart cytochrome c was expressed in E. coli during growth on isotopically enriched minimal media. Complete resonance assignments of both the diamagnetic reduced (spin zero) and paramagnetic oxidized (spin ½) states of the protein were obtained using standard triple resonance and total correlation spectroscopy using the previously determined 1H chemical shifts of the wild‐type protein as a guide. The correspondence of chemical shifts between the wild type and the mutant protein is excellent, indicating that they have nearly identical structures. The expanded library of chemical shifts for both redox states in both proteins allowed the refinement of the electron spin g‐tensor of the oxidized states. The g‐tensors of the oxidized states of the wild‐type and [H26N, H33N] mutant proteins are closely similar, indicating that the subtle details of the ligand fields are nearly identical. The refined g‐tensors were then used to probe for redox‐dependent structure change in the two proteins.
Opposing effects of phosphoenolpyruvate and pyruvate with Mg2+ on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli
Authors: Dimitrova Mariana N. , Peterkofsky Alan , Ginsburg Ann 
Published: August 2003

AbstractThe activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer‐dimer equilibrium where a Mg2+‐dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active‐site His189, substrate‐binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg2+) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20°C, 5 mM pyruvate (with 2 mM Mg2+) has the opposite effect. A correlation between the coupling of N‐ and C‐terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N‐ and C‐terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg2+ is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 108 to < 5 × 105 or 3 × 107 M−1, respectively. Incubation of the wild‐type, dephospho‐enzyme I with the transition‐state analog phosphonopyruvate and 2 mM Mg2+ also increases domain coupling and the dimerization constant ∼42‐fold. With 2 mM Mg2+ at 15–25°C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with KA′ ≈ 106 M−1 (ΔG′ = −8.05 ± 0.05 kcal/mole and ΔH = +3.9 kcal/mole at 20°C); ΔCp = −0.33 kcal K−1 mole−1. The binding of PEP to EI(H189A) is synergistic with that of Mg2+. Thus, physiological concentrations of PEP and Mg2+ increase, whereas pyruvate and Mg2+ decrease the amount of dimeric, active, dephospho‐enzyme I.
Rationally selected basis proteins: A new approach to selecting proteins for spectroscopic secondary structure analysis
Authors: Oberg Keith A. , Ruysschaert Jean‐Marie , Goormaghtigh Erik 
Published: August 2003

AbstractProtein basis sets have been extensively used as reference data for the determination of protein structure with optical methods such as circular dichroism and infrared spectroscopies. We have taken a new approach to basis protein selection by utilizing three crystal structure classification databases: CATH, SCOP, and PDB_SELECT. Through the use of the information available in these and other online resources, we identified 115 commercially available proteins as potential basis set candidates. By carefully screening the quality of the crystal structures and commercial protein preparations, we obtained a final set of 50 rationally selected proteins (RaSP50) that has been optimized for use in spectroscopic protein structure determination studies. These proteins span the full range of known protein folds as well as α‐helix and β‐sheet contents, and they represent a more comprehensive variety of fold types than any previous reference set. This report includes a detailed presentation of the reasoning behind the rational protein selection process, a description of the properties of the RaSP50 set, and a discussion of the types of structural and spectral variations that are represented in the set.
Homology modeling provides insights into the binding mode of the PAAD/DAPIN/pyrin domain, a fourth member of the CARD/DD/DED domain family
Authors: Liu Tong , Rojas Ana , Ye Yuzhen , Godzik Adam 
Published: August 2003

AbstractThe PAAD/DAPIN/pyrin domain is the fourth member of the death domain superfamily, but unlike other members of this family, it is involved not only in apoptosis but also in innate immunity and several other processes. We have identified 40 PAAD domain‐containing proteins by extensively searching the genomes of higher eukaryotes and viruses. Phylogenetic analyses suggest that there are five categories of PAAD domains that correlate with the domain architecture of the entire proteins. Homology models built on CARD and DD structures identified functionally important residues by studying conservation patterns on the surface of the models. Surface maps of each subfamily show different distributions of these residues, suggesting that domains from different subfamilies do not interact with each other, forming independent regulatory networks. Helix3 of PAAD is predicted to be critical for dimerization. Multiple alignment analysis and modeling suggest that it may be partly disordered, following a new paradigm for interaction proteins that are stabilized by protein–protein interactions.
Discrete restraint‐based protein modeling and the Cα‐trace problem
Authors: DePristo Mark A. , de Bakker Paul I.W. , Shetty Reshma P. , Blundell Tom L. 
Published: August 2003

AbstractWe present a novel de novo method to generate protein models from sparse, discretized restraints on the conformation of the main chain and side chain atoms. We focus on Cα‐trace generation, the problem of constructing an accurate and complete model from approximate knowledge of the positions of the Cα atoms and, in some cases, the side chain centroids. Spatial restraints on the Cα atoms and side chain centroids are supplemented by constraints on main chain geometry, φ/ξ angles, rotameric side chain conformations, and inter‐atomic separations derived from analyses of known protein structures. A novel conformational search algorithm, combining features of tree‐search and genetic algorithms, generates models consistent with these restraints by propensity‐weighted dihedral angle sampling. Models with ideal geometry, good φ/ξ angles, and no inter‐atomic overlaps are produced with 0.8 Å main chain and, with side chain centroid restraints, 1.0 Å all‐atom root‐mean‐square deviation (RMSD) from the crystal structure over a diverse set of target proteins. The mean model derived from 50 independently generated models is closer to the crystal structure than any individual model, with 0.5 Å main chain RMSD under only Cα restraints and 0.7 Å all‐atom RMSD under both Cα and centroid restraints. The method is insensitive to randomly distributed errors of up to 4 Å in the Cα restraints. The conformational search algorithm is efficient, with computational cost increasing linearly with protein size. Issues relating to decoy set generation, experimental structure determination, efficiency of conformational sampling, and homology modeling are discussed.
Refolding out of guanidine hydrochloride is an effective approach for high‐throughput structural studies of small proteins
Authors: Maxwell Karen L. , Bona Diane , Liu Chengsong , Arrowsmith Cheryl H. , Edwards Aled M. 
Published: August 2003

AbstractLow in vivo solubility of recombinant proteins expressed in Escherichia coli can seriously hinder the purification of structural samples for large‐scale proteomic NMR and X‐ray crystallography studies. Previous results from our laboratory have shown that up to one half of all bacterial and archaeal proteins are insoluble when overexpressed in E. coli. Although a number of strategies may be used to increase in vivo protein solubility, there are no generally applicable methods, and the expression of each insoluble recombinant protein must be individually optimized. For this reason, we have tested a generic denaturation/refolding protein purification procedure to assess the number of structural samples that could be generated by using this methodology. Our results show that a denaturation/refolding protocol is appropriate for many small proteins (≤18 kD) that are normally soluble in vivo. In addition, refolding the purified proteins by using dialysis against a single buffer allowed us to obtain soluble protein samples of 58% of small proteins that were found in the insoluble fraction in vivo, and 10% of the initial number of proteins provided good heteronuclear single quantum coherence (HSQC) NMR spectra. We conclude that a denaturation/refolding protocol is an efficient way to generate structural samples for high‐throughput studies of small proteins.
Lipid–binding proteins: Structure of the phospholipid ligands
Authors: Marsh Derek 
Published: August 2003

AbstractDihedral angles are evaluated for the phospholipid ligands of the lipid‐binding proteins found in the Protein Data Base (PDB). Phospholipid structures occur with a trans C1–C2 configuration of the glycerol backbone and oppositely extended chains, in addition to the gauche C1–C2 rotamers found in membranes. Headgroup conformations are not restricted to the single bent‐down configuration and gauche–gauche configuration of the phosphodiester that is found in phospholipid crystals. Additionally, fully extended headgroups and orientations directed away from the lipid chains are found for phospholipids in the protein binding pockets. On average, the hydrocarbon chains of the protein‐bound lipids are conformationally more disordered than in fluid bilayer membranes. However, much of this configurational disorder arises from energetically disallowed skew conformations. This suggests a configurational heterogeneity in the lipids at a single binding site: Eclipsed conformations occur also in some lipid headgroups and glycerol backbones. Stereochemical violations appear for some of the ester carboxyl groups of the protein‐bound phospholipids in the PDB, and two glycerol backbones have the incorrect enantiomeric configuration.
Evidence from panel unit root and cointegration tests that the Environmental Kuznets Curve does not exist
Authors: Perman Roger , Stern David I. 
Published: August 2003

The Environmental Kuznets Curve (EKC) hypothesis – an inverted U‐shape relation between various indicators of environmental degradation and income per capita – has become one of the ‘stylised facts’ of environmental and resource economics. This is despite considerable criticism on both theoretical and empirical grounds. Cointegration analysis can be used to test the validity of such stylised facts when the data involved contain stochastic trends. In the present paper, we use cointegration analysis to test the EKC hypothesis using a panel dataset of sulfur emissions and GDP data for 74 countries over a span of 31 years. We find that the data is stochastically trending in the time‐series dimension. Given this, and interpreting the EKC as a long run equilibrium relationship, support for the hypothesis requires that an appropriate model cointegrates and that sulfur emissions are a concave function of income. Individual and panel cointegration tests cast doubt on the general applicability of the hypothesised relationship. Even when we find cointegration, many of the relationships for individual countries are not concave. The results show that the EKC is a problematic concept, at least in the case of sulfur emissions.
Hazardous Facility Siting When Cost Information Is Private: An Application of Multidimensional Mechanism Design
Authors: Waehrer Keith 
Published: August 2003

AbstractThe siting of hazardous facilities often involves externalities that extend beyond the border of the community selected as a site. Thus, the private information of each community is potentially a vector of costs comprising a cost for each of the possible sites. I characterize the conditions for the existence of a direct mechanism that is incentive compatible, individually rational, and budget balancing. Incentive compatibility implies a pattern of compensation payments that often conflicts with compensation policy goals. When nonparticipating communities cannot block the siting of the facility, it will often be possible to implement siting policies with a balanced budget.
Modeling alternative zoning strategies in forest management
Authors: Krcmar Emina , Vertinsky Ilan , Van Kooten G. Cornelis 
Published: August 2003

AbstractTo satisfy public demands for environmental values, forest companies are facing the prospect of a reduction in wood supply and increases in costs. Some Canadian provincial governments have proposed intensifying silviculture in special zones dedicated to timber production as the means for pushing out the forest possibility frontiers. In this paper, we compare the traditional two‐zone land allocation framework which includes ecological reserves and integrated forest management zones with the triad — a three‐zone scheme which adds a zone dedicated to intensive timber production. We compare the solutions of the mixed‐integer linear programs formulated under both land‐allocation frameworks. We explore through sensitivity analysis the conditions under which the triad regime can offset the impact on timber production from increased environmental demands. We show that under the realistic conditions characteristic to Coastal British Columbia, higher environmental demands may be satisfied under the triad regime without increasing the financial burdens on the industry or reducing its wood supply. This occurs, however, only if regulatory constraints in timber production zone are flexible.
Functional Analysis of Mosquito-Borne Flavivirus Conserved Sequence Elements within 3′ Untranslated Region of West Nile Virus by Use of a Reporting Replicon That Differentiates between Viral Translation and RNA Replication
Authors: Lo Michael K. , Tilgner Mark , Bernard Kristen A. , Shi Pei-Yong 
Published: August 2003

ABSTRACT

We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A
Renilla
luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NS5mt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3′ untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5′ region of the capsid gene (5′CS) abolished RNA replication. Restoration of the 5′CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5′CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication.
SR-Related Protein TAXREB803/SRL300 Is an Important Cellular Factor for the Transactivational Function of Human T-Cell Lymphotropic Virus Type 1 Tax
Authors: Youn Hwang-Geum , Matsumoto Jun , Tanaka Yuetsu , Shimotohno Kunitada 
Published: August 2003

ABSTRACT
Expression of the human T-cell lymphotropic virus type 1 (HTLV-1) genes is transcriptionally activated by the cognate oncoprotein Tax which enhances the binding of the cyclin AMP-responsive element binding protein (CREB) to the Tax responsive element (TxRE) located in its long terminal repeat (LTR). TxRE is highly homologous to the cyclic AMP-responsive element (CRE) except for the GC-rich sequence flanking the CRE. We cloned the cDNA for a cellular factor, TAXREB803, of which the DNA-binding domain bound to TxRE and the binding was dependent on the 3′ GC-rich sequence in TxRE. TAXREB803 is an SR-related protein composed of 2,752 amino acids including numerous arginine/serine (RS) motifs. TAXREB803 enhanced both the Tax dependent transcription and the CREB binding to TxRE in cooperation with Tax. The interaction of TAXREB803 and Tax was detected by coimmunoprecipitation assays as well as by indirect immunofluorescence assays. Significantly, Tax transactivation for the HTLV-1 LTR decreased dramatically when the expression level of the endogenous TAXREB803 was suppressed by the small interfering RNA. These results suggest that TAXREB803 functions as a transcriptional coactivator for Tax and plays a critical role in the expression of HTLV-1 genes.
Theoretical Design of a Gene Therapy To Prevent AIDS but Not Human Immunodeficiency Virus Type 1 Infection
Authors: Weinberger Leor S. , Schaffer David V. , Arkin Adam P. 
Published: August 2003

ABSTRACTRecent reports confirm that, due to the presence of long-lived, latently infected cell populations, eradication of human immunodeficiency virus type 1 (HIV-1) from infected patients by using antiretroviral drugs will be exceedingly difficult. An alternative to virus eradication may be to use gene therapy to induce a pseudo-latent state in virus-producing cells, thus transforming HIV-1 into a lifelong, but manageable, virus. Conditionally replicating HIV-1 (crHIV-1) gene therapy vectors provide an avenue for subduing HIV-1 expression in infected cells (by creating a parasite, crHIV-1, of the parasite HIV-1), potentially reducing the HIV-1 set point and delaying AIDS onset. Development of crHIV-1 vectors has proceeded in vitro, but the requirements for a crHIV-1 vector to proliferate and persist in vivo have not been explored. We expand a widely accepted mathematical model of HIV-1 in vivo dynamics to include a crHIV-1 gene therapy virus and derive a simple criterion for designing crHIV-1 viruses that will persist in vivo. The model introduces only two new parameters—HIV-1 inhibition and crHIV-1 production—and both can be experimentally engineered and controlled. Analysis demonstrates that crHIV-1 gene therapy can indefinitely reduce HIV-1 set point to levels comparable to those achieved with highly active antiretroviral therapy, provided crHIV-1 production is more efficient than HIV-1. Paradoxically, highly efficient therapeutic inhibition of HIV-1 was found to be disadvantageous. Thus, the field may benefit by shifting the search for more potent antiviral genes toward engineering optimized therapy viruses that package ultraefficiently while downregulating viral production moderately.