Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns.

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals-Fe(II), Cu(II) and Ni(II)-on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis.

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.

Structure Analysis of Proteins and Peptides by Difference Circular Dichroism Spectroscopy.

Difference circular dichroism (CD) spectroscopy was used here to characterize changes in structure of flexible peptides upon altering their environments. Environmental changes were introduced by binding to a large target structure, temperature shift (or concentration increase) or so-called membrane-mimicking solvents. The first case involved binding of a largely disordered peptide to its target structure associated with chromatin remodeling, leading to a transition into a highly helical structure. The second example was a short 8HD (His-Asp) repeat peptide that can bind metal ions. Both Zn and Ni at µM concentrations resulted in different type of changes in secondary structure, suggesting that these metal ions provide different environments for the peptide to assume unique secondary structures. The third case is related to a few short neuroprotective peptides that were largely disordered in aqueous solution. Increased temperature resulted in induction of significant, though small, ß-sheet structures. Last example was the induction of non-helical structures for short neuroprotective peptides by membrane-mimicking solvents, including trifluoroethanol, dodecylphosphocholine and sodium dodecylsulfate. While these agents are known to induce α-helix, none of the neuropeptides underwent transition to a typical helical structure. However, trifluoroethanol did induce α-helix for the first peptide involved in chromatin remodeling described above in the first example.

Insight into the protein salting-in mechanism of arginine, magnesium chloride and ethylene glycol: Solvent interaction with aromatic solutes.

Key factors in the salting-in effects on proteins of additives are their interactions with aromatic groups. We studied the interaction of four aromatic solutes, benzyl alcohol (BA), phenol, 4-hydroxybenzyl alcohol (4-HBA) and methyl gallate (MG), with different salting-in additives, arginine hydrochloride (ArgHCl), magnesium chloride (MgCl2), ethylene glycol (EG), and guanidine hydrochloride (GdnHCl) using solubility measurements. We used sodium chloride (NaCl) as a control. MgCl2 decreased the solubility of the four aromatic solutes with weak solute dependence. In contrast, ArgHCl, GdnHCl, and EG increased the solubility of four aromatic solutes with a similar solute dependence. Their salting-in effects were weaker on BA and 4-HBA and stronger on phenol and MG. These results indicate that attached groups alter the aromatic properties, affecting the interactions between the benzene ring and these three additives. More importantly, the observed results demonstrate that the salting-in mechanism is different between MgCl2, EG and ArgHCl, which should play a role in their effects on protein solubility.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis.

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

Structural insights into assembly and function of the RSC chromatin remodeling complex.

SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.

The Glucagon-Like Peptide 2 Analog Teduglutide Reversibly Associates to Form Pentamers.

Glucagon-like peptide 1 and 2 and their analog peptide therapeutics are known to reversibly associate to form oligomers. Here we report the association properties of the glucagon-like peptide 2 analog teduglutide at concentrations up to ∼15 mg/mL. Both sedimentation equilibrium (SE-AUC) and sedimentation velocity (SV-AUC) show that teduglutide dissociates completely to monomers below 0.1 mg/mL. SE-AUC shows that the apparent weight-average molar mass increases substantially between 0.1 and 1 mg/mL, reaching a maximum of ∼14.5 kDa (∼3.9-mer) near 2 mg/mL, and then falling at higher concentrations because of strong solution nonideality effects (highly positive second virial coefficient). Circular dichroism spectra over the range from 0.1 to 2 mg/mL show that self-association is accompanied by significant increases in alpha-helix content, and that the associated state has a distinct tertiary structure. The SV-AUC data up to 2.2 mg/mL are fitted fairly well by an ideal rapidly reversible monomer-pentamer association. The SE-AUC modeling included thermodynamic nonideality effects. SE-AUC data up to ∼15 mg/mL imply a monomer-pentamer association at lower concentrations, but the pentamers also appear to weakly associate to form decamers. These results illustrate the importance of directly modeling the solution nonideality effects, which if neglected would lead to an incorrect preferred stoichiometry.

A Small-Molecule Pan-Id Antagonist Inhibits Pathologic Ocular Neovascularization.

Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.

Agarose native gel electrophoresis of proteins.

We have developed an agarose-based native gel electrophoresis system that works for both acidic and basic proteins using histidine-MES buffer. This electrophoresis can be done in a flat-bed mode or a vertical mode. While in the flat-bed mode both acidic and basic proteins can be simultaneously analyzed, the vertical gel can only be used for either protein. We have observed that while the migration of acidic bovine serum albumin (BSA) was independent of the buffer concentration, the behavior of basic lysozyme was greatly improved at higher buffer concentration, e.g., 100 mM histidine-100 mM MES. With this buffer system, BSA, lysozyme and chymotrypsin showed expected band mobility and Adeno associated virus particle and bovine gamma globulin showed apparent basic nature of the surface properties.

Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution.

Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0 M NaCl at 25 °C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution.

Cys139Ser mutation in dimeric nucleoside diphosphate kinase generates catalytically competent monomer.

Nucleoside diphosphate kinase from a moderate halophile Halomonas sp. 593 (HaNDK) is dimer, while NDK from different origins has been shown to assemble into hexamer, or tetramer. Similar to HaNDK, halophilic NDK from Chromohalobacter salexigens DSM3043 (CsNDK) formed dimeric structure. Cysteine139 conserved between HaNDK and CsNDK is located in the monomer/monomer interface. Substitution of Cys139 for Ser caused dissociation of dimeric CsNDK into monomer in Tris buffer, as determined by field flow fractionation technique. Circular dichroism (CD) profile of the mutant CsNDK was nearly identical to the wild type CsNDK: however, the mutant CsNDK became more susceptible to "endproteinase GluC" cleavage, which could be suppressed by an NDK substrate, ATP. The monomer was enzymatically active, although it is possible that active structure is dimer in the presence of substrate.

Inactive C8A­humanin analog is as stable as a potent S14G­humanin analog.

We have previously shown that the structural stability of humanin (HN), a neuroprotective peptide ligand, is one of the attributes to the observed activity differences between HN analogs. It has been observed that the activity increased consecutively in the S7A­HN analog, the parent HN and the S14G­HN analog, consistent with the increased stability observed in that order. In the present study, the structure and stability of another inactive analog, C8A­HN, was measured, which has been revealed to have no neuroprotective activity similar to that of the S7A­HN analog and hence may have compromised stability. While all these analogs of HN demonstrated a similar disordered secondary structure in phosphate-buffered saline at 5˚C, as determined by circular dichroism spectroscopy, they revealed different structures at 37˚C. At 37˚C, less active HN and inactive S7A­HN revealed a structure with a valley at ~217 nm, indicating a conversion from the disordered structure to a ß­sheet. Such a conversion was largely irreversible. By contrast, C8A­HN and S14G­HN demonstrated a similar structure at 37˚C and at 5˚C and remained largely disordered. The observed small structural changes of the C8A­HN analog at 37˚C and its reversibility upon cooling do not support a hypothesis that the instability at 37˚C may have caused the reduced activity of this analog. Therefore an alternative explanation for its activity loss is required.

Structural characteristics of short peptides in solution.

Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or ß-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability.

Synergistic solubilization of porcine myosin in physiological salt solution by arginine.

Myosin is an important protein resource for food industries and has a bipolar filamentous structure that is composed of subfilaments that occur in vivo. It has been shown that a high ionic strength is required to prevent myosin from forming filamentous structures and to solubilize the protein in aqueous solution. In the presence of 100-200 mM NaCl, 50 mM arginine was more effective than other additives tested, including NaCl, in myosin solubilization. Before reaching equilibrium solubility, the myosin solution was initially supersaturated upon the dilution of a stock myosin solution in 1 M NaCl into the test solvents. Arginine slowed the process of equilibration and stabilized the supersaturated solution more effectively than other additives. No structural changes in myosin caused by arginine were observed, which indicated that arginine enhanced the solubility of myosin in a physiological salt solution without affecting the structure.

Amyloid fibril formation in vitro from halophilic metal binding protein: its high solubility and reversibility minimized formation of amorphous protein aggregations.

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine-rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His-tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low-pH harsh conditions, however, His-HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid-hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full-length His-HP when incubated with 10-20% 2,2,2-trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.

Structure of three Humanin peptides with different activities upon interaction with liposome.

We have recently shown that a 24 amino acid Humanin (HN) adopts an anti-parallel ß-sheet structure in the presence of a negatively charged 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and suggested a possibility that it interacts with lipid membranes and thereby exerts neuroprotective effects through the target cell surface receptors or the intracellular signaling molecules following membrane interaction events. The structures of two HN analogs, having either a S7A mutation or a S14G mutation, were examined under the identical conditions, as the S7A analog is inactive and the S14G analog is 1000-fold more active than the wild type HN. These analogs showed a secondary structure indistinguishable from the structure of HN in the presence of DOPG liposome, while unrelated peptides were disordered with and without DOPG. It thus appeared that HN and the analogs, regardless of the biological activities, have an ability to interact with DOPG liposome and form an anti-parallel ß-sheet structure. While the wild type HN and the S7A and S14G analogs were largely disordered in buffer, the S14G analog showed greater stability as a disordered structure in the buffer at a physiological temperature, suggesting that it maintains the disordered structure presumably required for the interaction with the DOPG liposome and thereby greater neuroprotective activity.

A novel protein refolding system using lauroyl-l-glutamate as a solubilizing detergent and arginine as a folding assisting agent.

More than 50 detergents, including acylated amino acid derivatives, were screened for their ability to solubilize and refold recombinant proteins expressed as inclusion bodies. Two model proteins, human interleukin-6 and microbial transglutaminase, were solubilized by these detergents and the solubilized proteins were rapidly diluted for testing their solubilization and refolding effectiveness. Long chain-acylated amino acid derivatives having dicarboxylic acid moieties were found to be superior to others under the conditions tested. In particular, lauroyl-l-glutamate (C12-l-Glu) showed the highest recovery of the native proteins. The effectiveness of dilution refolding was greatly improved by adding aggregation suppressive arginine into the refolding solvents. To gain understanding how this detergent works, interactions between detergents and proteins were examined using spectroscopic and native gel electrophoretic analyses, showing ideal properties for C12-l-Glu as a solubilzing agent, i.e. highly reversible nature of the detergent binding to the model globular proteins and of the conformational changes. These properties most likely have contributed to the effective protein solubilzation and refolding of inclusion bodies using C12-l-Glu and arginine.

Non-denaturing solubilization of inclusion bodies.

It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1alpha, interferon-gamma, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturating solubilization of some of these proteins we have tested.

Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site.

The binding of atrial natriuretic peptide (ANP) to its receptor requires chloride, and it is chloride concentration dependent. The extracellular domain (ECD) of the ANP receptor (ANPR) contains a chloride near the ANP-binding site, suggesting a possible regulatory role. The bound chloride, however, is completely buried in the polypeptide fold, and its functional role has remained unclear. Here, we have confirmed that chloride is necessary for ANP binding to the recombinant ECD or the full-length ANPR expressed in CHO cells. ECD without chloride (ECD(-)) did not bind ANP. Its binding activity was fully restored by bromide or chloride addition. A new X-ray structure of the bromide-bound ECD is essentially identical to that of the chloride-bound ECD. Furthermore, bromide atoms are localized at the same positions as chloride atoms both in the apo and in the ANP-bound structures, indicating exchangeable and reversible halide binding. Far-UV CD and thermal unfolding data show that ECD(-) largely retains the native structure. Sedimentation equilibrium in the absence of chloride shows that ECD(-) forms a strongly associated dimer, possibly preventing the structural rearrangement of the two monomers that is necessary for ANP binding. The primary and tertiary structures of the chloride-binding site in ANPR are highly conserved among receptor-guanylate cyclases and metabotropic glutamate receptors. The chloride-dependent ANP binding, reversible chloride binding, and the highly conserved chloride-binding site motif suggest a regulatory role for the receptor bound chloride. Chloride-dependent regulation of ANPR may operate in the kidney, modulating ANP-induced natriuresis.

Antiviral and virucidal activities of natural products.

Virus infection is one of the major threats to human health and can be avoided by minimizing exposure to infectious viruses. Viral clearance of pharmaceutical products and sanitization of skin and mucosal surfaces would reduce such exposures. Even with such care, virus infection does occur, requiring effective treatments by antiviral or virucidal agents. Natural products, in particular ingredients of foods and drinks we normally consume or metabolites present in human body at low concentrations, would have advantage over synthetic drugs as antiviral agents for safety concerns. For this reason, we have been studying natural products for their effects on virus inactivation and growth. Such natural products, which we have been focusing, include gallate derivatives, caffeine present in coffee, caffeic acid present in coffee and various fruits, ascorbic and dehydroascorbic acids and a cell metabolite, arginine. Here we will review our work on antiviral and virucidal activities of these compounds and the mechanism of their antiviral and virucidal effects.