Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

Partial Least-Squares Functional Mode Analysis: Application to the Membrane Proteins AQP1, Aqy1, and CLC-ec1

We introduce an approach based on the recently introduced functional mode analysis to identify collective modes of internal dynamics that maximally correlate to an external order parameter of functional interest. Input structural data can be either experimentally determined structure ensembles or simulated ensembles, such as molecular dynamics trajectories. Partial least-squares regression is shown to yield a robust solution to the multidimensional optimization problem, with a minimal and controllable risk of overfitting, as shown by extensive cross-validation. Several examples illustrate that the partial least-squares-based functional mode analysis successfully reveals the collective dynamics underlying the fluctuations in selected functional order parameters. Applications to T4 lysozyme, the Trp-cage, the aquaporin channels Aqy1 and hAQP1, and the CLC-ec1 chloride antiporter are presented in which the active site geometry, the hydrophobic solvent-accessible surface, channel gating dynamics, water permeability (p(f)), and a dihedral angle are defined as functional order parameters. The Aqy1 case reveals a gating mechanism that connects the inner channel gating residues with the protein surface, thereby providing an explanation of how the membrane may affect the channel. hAQP1 shows how the p(f) correlates with structural changes around the aromatic/arginine region of the pore. The CLC-ec1 application shows how local motions of the gating Glu(148) couple to a collective motion that affects ion affinity in the pore.     

Structural insights into Pot1-ssDNA,Pot1-Tpz1 and Tpz1-Ccq1 Interactions within fission yeast shelterin complex

The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1_OB1-GGTTAC and Pot1_OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1_DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1_DBD-single-stranded-DNA, Pot1_372-555-Tpz1_185-212 and Tpz1_425-470-Ccq1_123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1_DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3’ ss telomeric overhang.     

Multistep Aggregation Pathway of Human Interleukin-1 Receptor Antagonist: Kinetic, Structural, and Morphological Characterization

The complex, multistep aggregation kinetic and structural behavior of human recombinant interleukin-1 receptor antagonist (IL-1ra) was revealed and characterized by spectral probes and techniques. At a certain range of protein concentration (12-27 mg/mL) and temperature (44-48°C), two sequential aggregation kinetic transitions emerge, where the second transition is preceded by a lag phase and is associated with the main portion of the aggregated protein. Each kinetic transition is linked to a different type of aggregate population, referred to as type I and type II. The aggregate populations, isolated at a series of time points and analyzed by Fourier-transform infrared spectroscopy, show consecutive protein structural changes, from intramolecular (type I) to intermolecular (type II) β-sheet formation. The early type I protein spectral change resembles that seen for IL-1ra in the crystalline state. Moreover, Fourier-transform infrared data demonstrate that type I protein assembly alone can undergo a structural rearrangement and, consequently, convert to the type II aggregate. The aggregated protein structural changes are accompanied by the aggregate morphological changes, leading to a well-defined population of interacting spheres, as detected by scanning electron microscopy. A nucleation-driven IL-1ra aggregation pathway is proposed, and assumes two major activation energy barriers, where the second barrier is associated with the type I → type II aggregate structural rearrangement that, in turn, serves as a pseudonucleus triggering the second kinetic event.     

Crystal structure of mSULT1D1, a mouse catecholamine sulfotransferase

In mammals, sulfonation as mediated by specific cytosolic sulfotransferases (SULTs) plays an important role in the homeostasis of dopamine and other catecholamines. To gain insight into the structural basis for dopamine recognition/binding, we determined the crystal structure of a mouse dopamine-sulfating SULT, mouse SULT1D1 (mSULT1D1). Data obtained indicated that mSULT1D1 comprises of a single α/β domain with a five-stranded parallel β-sheet. In contrast to the structure of the human SULT1A3 (hSULT1A3)-dopamine complex previously reported, molecular modeling and mutational analysis revealed that a water molecule plays a critical role in the recognition of the amine group of dopamine by mSULT1D1. These results imply differences in substrate binding between dopamine-sulfating SULTs from different species.     

Expression,Purification and Low-Resolution Structure of Human Vitamin C Transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.     

Solution structure of PcFK1, a spider peptide active against Plasmodium falciparum

Psalmopeotoxin I (PcFK1) is a 33-amino-acid residue peptide isolated from the venom of the tarantula Psalmopoeus cambridgei. It has been recently shown to possess strong antiplasmodial activity against the intra-erythrocyte stage of Plasmodium falciparum in vitro. Although the molecular target for PcFK1 is not yet determined, this peptide does not lyse erythrocytes, is not cytotoxic to nucleated mammalian cells, and does not inhibit neuromuscular function. We investigated the structural properties of PcFK1 to help understand the unique mechanism of action of this peptide and to enhance its utility as a lead compound for rational development of new antimalarial drugs. In this paper, we have determined the three-dimensional solution structure by (1)H two-dimensional NMR means of recombinant PcFK1, which is shown to belong to the ICK structural superfamily with structural determinants common to several neurotoxins acting as ion channels effectors.     

Computational studies of CXCR1, the receptor of IL-8/CXCL8, using molecular dynamics and electrostatics

The three-dimensional structure of IL-8/CXCL8 has been previously determined using NMR spectroscopy and X-ray crystallography, but the structure of the receptors for this chemokine has not been determined experimentally. We present here the development of a model for the structure of the IL-8/CXCL8 receptor CXCR1, using a combination of homology modeling and a molecular dynamics simulation. Based on this model, we discuss the analysis of structural, dynamic, and physicochemical properties of CXCR1. We focused on the role of pairwise ionic interactions in local structural stability of CXCR1 and the role of electrostatic potentials in recognition of CXCR1 with IL-8/CXCL8. We have performed theoretical mutations of six charged amino acids in CXCR1, which abolish binding as suggested by earlier experimental data, to shed light on the effect of charge on association ability. We propose that the observed loss of binding in the six CXCR1 mutants is owed to loss of local structural stability, rather than hindrance of the recognition process because of changes in the overall electrostatic properties of the receptor. Based on further structural analysis, we propose some mutations of charged residues involving ion pairs in different elements of transmembrane helices and extracellular loops, which are expected to alter the local structure and possibly affect binding.     

Studies of 1-deoxy-D-fructose, 1-deoxy-D-glucitol, and 1-deoxy-D-minnitol as antimetabolites.

1-Deoxy-D-fructose was synthesized in 27% yield from D-glucosamine in a three-step procedure involving Raney nickel desulfurization and oxidative deamination with 3,5-di-tert-butyl- 1,2-benzoquinone applied to appropriate intermediates. 1-Deoxyfructose and its reduction products, 1-deoxyglucitol and 1-deoxymannitol, were tested as substrates and antimetabolites. For sheep liver glucitol dehydrogenase, the Km is 53 mM for 1-deoxymannitol, were tested as substrates and antimetabolites. For sheep liver glucitol dehydrogenase, the Km is 53 mM for 1-deoxyglucitol and 89 mM for 1-deoxymannitol with maximal velocities 33 and 18%, respectively, of that with glucitol as substrate. These results require substantial revision of the long-accepted polyol substrate structural requirements for this enzyme which have been reported to include a 1-hydroxy group and a cis-2,4-dihydroxy configuration. Km is 614 and 280 mM for yeast and muscle hexokinases, respectively, acting on 1-deoxyfructose; maximal velocities are 2 and 5% of those obtained with fructose. 1-Deoxyfructose 6-phosphate is a competitive inhibitor of phosphoglucose isomerase with a Ki of 1.1 mM; this is about the same as Km for the natural substrates. It is also an effective inhibitor of phosphofructokinase but does not alter the cooperativity of the enzyme interaction with fructose 6-phosphate nor exhibit cooperativity in its own interaction therewith. These results suggest that the 1-hydroxy group is not crucial for binding but does play a role in the cooperative interactions of this allosteric protein. At equivalent concentrations, 1-deoxyfructose is somewhat better than 2-deoxyglucose as an inhibitor of erythrocyte glycolysis; the 1-deoxypolyols are ineffective. All three 1-deoxy compounds are readily, though incompletely, absorbed from the intestine of mice; most of the absorbed dose appears in the urine unchanged within 24 h. Whether given by oral or intraperitoneal routes, 2 to 6% of administered deoxypolyol or deoxyketose appears in the urine as ketose or polyol, respectively. No acute toxic effects or growth retardation are noted for any of the 1-deoxy analogues when given to mice at levels where 2-deoxyglucose has such effects. The properties of these 1-deoxy sugar analogues recommend them for further studies of enzyme mechanisms, for metabolic studies, and for testing as therapeutic agents against such organisms as certain mammalian parasites with heavy reliance on glycolysis.     

Synthesis of new 1-alkyl-, 1-benzyl-, and 1-aryloxyethyl-substituted 4,5-dichloroimidazoles and their antimicrobial,protistocidal, and fungistatic properties

Previously unknown 1-alkyl-, 1-benzyl-, and 1-aryloxyethylderivatives of dichloroimidazoles and products of their structural transformation were synthesized from 4,5-dichloroimidazole or 2-methyl-4,5- dichloroimidazole using alkyl, benzyl or aryloxyethyl halides. These N-substituted compounds were shown to have a weak antibacterial activity against some pathogenic gram-positive and gram-negative bacteria (Staphylococcus aureus and Escherichia coli). At the same time, some of the obtained compounds demonstrated a significant protistocidal activity against Colpoda steinii, which can exceed in strength the activity of clinically used veterinary drug Baycox. Moreover, these compounds showed a pronounced fungistatic effect.     

The Russian gene pool. Genogeography of erythrocyte genetic markers (ACP1, PGM1, ESD, GLO1, 6-PGD)

The study continues the series of works on the Russian gene pool. Gene geographic analysis of five erythrocytic gene markers best studied in the Russian population (ACP1, PGM1, ESD, GLO1, and 6-PGD) has been performed. Gene-geographic electronic maps have been constructed for 13 alleles of these loci and their correlations with geographic latitude and longitude. For all maps, statistical characteristics are presented, including the variation range and mean gene frequencies, partial and multiple correlations with latitude and longitude, and parameters of heterozygosity and interpopulation diversity. The maps of eight alleles (ACP1*A, ACP1*C, PGM1*2+, PGM1*2-, PGM1*1-, ESD*1, GLO1*1, and PGD*C) are shown and analyzed in detail. The genetic relief and structural elements of the maps are compared with the ecumenical trends, main variation patterns of these genes in northern Eurasia, and genetic characteristics of the indigenous populations of the Urals and Europe.     

The RCC1 superfamily: from genes, to function, to disease

The Regulator of Chromosome Condensation 1 (RCC1) was identified over 20 years ago as a critical cell cycle regulator. By analyzing its amino acid sequence, RCC1 was found to consist of seven homologous repeats of 51-68 amino acid residues, which were later shown to adopt a seven-bladed beta-propeller fold. Since the initial identification of RCC1, a number of proteins have been discovered that contain one or more RCC1-like domains (RLDs). As we show here, these RCC1 superfamily proteins can be subdivided in five subgroups based on structural criteria. In recent years, a number of studies have been published regarding the functions of RCC1 superfamily proteins. From these studies, the emerging picture is that the RLD is a versatile domain which may perform many different functions, including guanine nucleotide exchange on small GTP-binding proteins, enzyme inhibition or interaction with proteins and lipids. Here, we review the available structural and functional data on RCC1 superfamily members, paying special attention to the human proteins and their involvement in disease.     

Obscurin-like 1, OBSL1, is a novel cytoskeletal protein related to obscurin

Cytoskeletal adaptor proteins serve vital functions in linking the internal cytoskeleton of cells to the cell membrane, particularly at sites of cell-cell and cell-matrix interactions. The importance of these adaptors to the structural integrity of the cell is evident from the number of clinical disease states attributable to defects in these networks. In the heart, defects in the cytoskeletal support system that surrounds and supports the myofibril result in dilated cardiomyopathy and congestive heart failure. In this study, we report the cloning and characterization of a novel cytoskeletal adaptor, obscurin-like 1 (OBSL1), which is closely related to obscurin, a giant structural protein required for sarcomere assembly. Multiple isoforms arise from alternative splicing, ranging in predicted molecular mass from 130 to 230 kDa. OBSL1 is located on human chromosome 2q35 within 100 kb of SPEG, another gene related to obscurin. It is expressed in a broad range of tissues and localizes to the intercalated discs, to the perinuclear region, and overlying the Z lines and M bands of adult rat cardiac myocytes. Further characterization of this novel cytoskeletal linker will have important implications for understanding the physical interactions that stabilize and support cell-matrix, cell-cell, and intracellular cytoskeletal connections.     

Analysis of quinolinequinone reactivity,cytotoxicity, and anti-HIV-1 properties

We have analyzed a set of quinolinequinones with respect to their reactivities, cytotoxicities, and anti-HIV-1 properties. Most of the quinolinequinones were reactive with glutathione, and several acted as sulfhydryl crosslinking agents. Quinolinequinones inhibited binding of the HIV-1 matrix protein to RNA to varying degrees, and several quinolinequinones showed the capacity to crosslink HIV-1 matrix proteins in vitro, and HIV-1 structural proteins in virus particles. Cytotoxicity assays yielded quinolinequinone CC₅₀ values in the low micromolar range, reducing the potential therapeutic value of these compounds. However, one compound, 6,7-dichloro-5,8-quinolinequinone potently inactivated HIV-1, suggesting that quinolinequinones may prove useful in the preparation of inactivated virus vaccines or for other virucidal purposes.     

Heteronuclear nuclear magnetic resonance assignments, structure and dynamics of SUMO-1, a human ubiquitin-like protein

The structure of a ubiquitin-like protein, small ubiquitin-related modifier-1 (SUMO-1), was earlier determined using homonuclear nuclear magnetic resonance (NMR) spectroscopy, since the spectral quality of the protein was not suitable for heteronuclear NMR data collection. In this study, a slightly different construct of the SUMO-1 gene was used for protein over-expression. The protein purified from this construct showed high spectral qualities, therefore, multi-dimensional heteronuclear NMR data for a dynamic study and structural determination were acquired. The structure of SUMO-1 obtained in this study differs in several respects from the structure obtained from homonuclear NMR data. Furthermore, structural differences were observed between the new SUMO-1 and ubiquitin structures. These differences may be important for SUMO-1-specific recognition in cells. Additionally, relaxation parameters indicate that SUMO-1 undergoes highly anisotropic tumbling in solution and that the long amino (N)-terminal sequence of SUMO-1 is highly dynamic with increasing flexibility towards the end.