Effects of L-arginine on solubilization and purification of plant membrane proteins

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins.     

The estimation of affinity constants for the binding of model peptides to DNA by equilibrium dialysis.

The binding of lysine model peptides of the type Lys-X-Lys, Lys-X-X-Lys and Lys-X-X-X-Lys (X = different aliphatic and aromatic amino acids) has been studied by equilibrium dialysis. It was shown that the strong electrostatic binding forces generated by protonated amino groups of lysine can be distinguished from the weak forces stemming from neutral and aromatic spacer amino acids. The overall binding strength of the lysine model peptides is modified by these weak binding forces and the apparent binding constants are influenced more by the hydrophobic character of the spacer amino acid side chains than by the chainlength of the spacers.     

Activity of amino acids as a function of their hydrophobicity in the process of the autolysis of theSaccharomyces cerevisiae yeast

The effect of amino acids on the process of autolysis of baker’s yeast was studied. It is shown that the addition of amino acids inhibits the increase in concentration of amino nitrogen during the process of autolysis. The effect of the inhibition is connected with the hydrophobicity of amino acids, the relationship being of the symbasis nature; it is especially obvious at high concentrations of the latter. The deviation from the symbasis as a result of the effect of low concentrations is the most typical for the highly hydrophobic amino acids (tyrosine, phenylalanine, isoleucine, tryptophan), which can be explained by their solubilization in lipid components of the cell. Hydrophilic glutamic acid suppresses both protease activity and nuclease activity of endoenzymes, which can be explained by its membranotrophic effect.     

Binding features of chloroplast fructose-1,6-bisphosphatase-thioredoxin interaction

It has been proposed that a hydrophobic groove surrounded by positively charged amino acids on thioredoxin (Trx) serves as the recognition and docking site for the interaction of Trx with target proteins. This model for Trx-protein interactions fits well with the Trx-mediated fructose-1,6-bisphosphatase (FBPase) activation, where a protruding negatively charged loop of FBPase would bind to this Trx groove, in a process involving both electrostatic and hydrophobic interactions. This model facilitates the prediction of Trx amino acid residues likely to be involved in enzyme binding. Site-directed mutagenesis of some of these amino acids, in conjunction with measurements of the FBPase activation capacity of the wild type and mutated Trxs, was used to check the model and provided evidence that lysine-70 and arginine-74 of pea Trx m play an essential role in FBPase binding. The binding parameters for the interaction between chloroplast FBPase and the wild type pea Trxs f and m, as well as mutated pea Trx m, determined by equilibrium dialysis in accordance with the Koshland-Nemethy-Filmer model of saturation kinetics, provided additional support for the role of these basic Trx residues in the interaction with FBPase. These data, in conjunction with the midpoint redox potential (E(m)) determinations of Trxs, support the hydrophobic groove model for the interaction between chloroplast FBPase and Trx. This model predicts that differences in the FBPase activation capacity of Trxs arise from their different binding abilities.     

Origin of the genetic code: A physical-chemical model of primitive codon assignments

Selective compartmentalization of amino acids and nucleotides according to their polarities is proposed as a physical-chemical model for the origin of the genetic code. Assumptions made in this hypothesis are: (1) an oil-slick covered the surface of the primitive ocean, constituents of which formed association colloids or micelles at the water-oil-air interfaces; (2) depending on the polarity of the media, these aggregates possessed hydrophilic and hydrophobic interiors where selective uptake of amino acids and nucleic acid constituents could take place; and 93) condensation and polymerization in the micellar phase were enhanced. According to the chromatographically observed polarities, for example, lysine and uridylate fall into the hydrophilic compartment, and phenylalanine and adenylate are enriched in the hydrophobic environment. These components could eventually be condensed to form a charged adaptor loop with an anticodon which is complementary to the presently valid codon. Only two groups of amino acids, hydrophilic and hydrophobic, were recognized by the primitive translation mechanism. Implications of this hypothesis for the further development of the genetic code is discussed. The catalytic power of micelles have been substantiated by successful synthesis of nucleotides under relatively mild conditions using thiophosphates as high energy phosphates.     

Hydrophobicity Density Profiles to Predict Thermal Stability Enhancement in Proteins

A hydrophobicity density is defined for a protein through its hydrophobicity tensor (similar to the inertia tensor), by using the Eisenberg hydrophobicity scale of the hydrophobic amino acids of a protein. This allows calculation of the radii of the corresponding hydrophobic ellipsoid of a protein and thus subsequently of its hydrophobic density. A hydrophobicity density profile is then obtained by simulating point mutations of each amino acid of a protein either to a high hydrophobicity value or to zero hydrophobicity. It is found that an increase in the hydrophobic density of the protein correlates with an increase of its mid-point transition temperature. From this profile it is possible to determine the amino acids or domain stretches in a protein that are most amenable to mutation in order to increase the thermal stability. The model is tested to predict the thermostabilisation effects of two mutations in a β-glucanase: M29G and M29F. This model is compared with other hydrophobicity-related profiles described by other authors.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Transforming activity of a 16-amino-acid segment of the bovine papillomavirus E5 protein linked to random sequences of hydrophobic amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the smallest transforming protein yet described. Previous results from our laboratory indicate that a hydrophobic core and specific carboxyl-terminal amino acids are required for the E5 protein to exert its transforming function. In this study, additional substitution mutations were generated in the E5 gene to determine the minimal amino acid sequence requirements for focus formation in mouse C127 cells. In most cases examined, substitution of the hydrophobic middle third of the E5 protein with unrelated hydrophobic sequences severely inhibited transforming activity. However, we have identified one hydrophobic amino acid sequence apparently unrelated to the wild-type one that can replace the middle third of the wild-type E5 protein without affecting the ability of the protein to stably transform cells or interact with cell membranes. Furthermore, a mutant E5 protein in which only the carboxyl-terminal 16 amino acids of the protein have been derived from E5 sequences retains transforming activity. Since several residues in the carboxyl-terminal portion of the E5 protein can be freely substituted with different amino acids (B. H. Horwitz, A. L. Burkhardt, R. Schlegel, and D. DiMaio, Mol. Cell. Biol. 8:4071-4078, 1988), the results reported here imply that much of the specific information necessary for cell transformation can be supplied by a subset of the carboxyl-terminal 16 amino acids of this protein.     

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic “hot spot” amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.     

The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein)

The amino-acid sequence of bovine myelin lipophilin (proteolipid apoprotein, Folch-protein) has been completed. Lipophilin is a 276 amino acid residues containing, extremely hydrophobic membrane protein with molecular mass 30,000 Da. The sequence determination was based on automated Edman degradation of four tryptophan and four cyanogen bromide fragments and of proteolytic peptides of complete lipophilin as well as the fragments obtained by chemical cleavage. Four additional sequences were determined which led to the completion of the primary structure. Lipophilin is esterified at threonine-198 by long chain fatty acids (palmitic, stearic and oleic acid). The attachment site has been established at the same threonine residue in three different peptides isolated from thermolysinolytic, papainolytic and chymotrypsinolytic hydrolysates. This threonine residue is part of a hydrophilic segment of lipophilin. The covalent fatty acyl bond is being discussed together with important structural and functional properties of this membrane protein which can be derived from sequence information. New separation and purification methods of hydrophobic and hydrophilic polypeptides for this sequence determination (fractional solubilization, silica gel exclusion, high-performance liquid chromatography) had to be elaborated as indispensable tools. They are generally applicable to the structural analysis of hydrophobic membrane proteins. Four long (26, 29, 40 and 36 residues) and one medium long (12 residues) hydrophobic segments are separated by four predominantly positively and one negatively charged hydrophilic segments. On the basis of structural data a model for the membrane integration of lipophilin is proposed.     

Role of hydrophobic clusters in the stability of alpha-helical coiled coils and their conversion to amyloid-like beta-sheets

We designed a library of short peptides using standard rules for coiled-coil assembly. Depending on the composition of amino acids in the non-interacting region of the coiled coil (positions b, c, and f) these peptides are able to convert from alpha-helical to beta-sheet secondary structure. This type of transition is observed in amyloid-like proteins and is a key feature associated with many types of neurodegenerative diseases. Studies on peptides that are 14 amino acids in length indicated that positioning hydrophobic amino acids at an f position within a heptad repeat accelerated the rate of conformational conversion as compared to that at a c position. We believe that this occurs because of the formation of a hydrophobic pocket that preferentially stabilizes beta-sheets over alpha-helices. This effect was also observed in longer 21 amino acid peptides. Our study shows that the relative rates of structural conversion correlate with the formation of a continuous three-amino-acid hydrophobic patch consisting of amino acids in the d, f, and a positions and not on the secondary structure propensities of the individual amino acids. The sequence-structure relationship observed in this study will be used to help understand the mechanism of amyloid fiber formation and design future coiled-coil and beta-sheet-forming peptide systems.     

Irreversible inhibition of hexosaminidase C by medium-chain monocarboxylic acids and Triton X-100

The neutral beta-N-acetylhexosaminidase (hexosaminidase C) from human brain was partially purified (separated from lysosomal beta-N-acetylhexosaminidases by chromatography on a Con A-Sepharose column). Hexosaminidase C was inhibited by medium-chain fatty acids (monocarboxylic acids with chain-length between C6 and C9), whereas shorter-chain monocarboxylic acids showed no inhibitory effect. Studies on the inhibition mechanism showed an irreversible and pH-dependent inhibition which progresses with time and which is not reversed by the removal of fatty acids (by Bio-Beads SM-2). Similar inhibitory effects were also obtained using Triton X-100 (but not with homologous alkylamines). These results suggest that the hexosaminidase C inactivation is related to the hydrophobic properties of the inhibitor which acts as a denaturing agent mainly at acidic pH. The possibility has been discussed that this inactivation effect of monocarboxylic acid on hexosaminidase C could constitute a molecular model of the toxicity of medium-chain-length fatty acids.     

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.     

A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains.

A liquid chromatographic stationary phase was prepared by covalently binding to the surface of microparticulate silica gel functionality (benzylsilane), which mimics the side chain of the amino acid phenylalanine. The chromatographic retentions of the N-acetyl C-(N'-methyl) amides of various hydrophobic and amphiphilic amino acids on this stationary phase were measured using an aqueous mobile phase. A retention order of Gly < Ala < Cys < Val < Met < Pro < Ile < Leu < Tyr < Phe < Trp is seen at room temperature. Chromatographic retentions were used to derive free energies of adsorption of the amino acid derivatives on the chromatographic support relative to that of the glycine derivative. The temperature dependencies of the retention of aromatic and aliphatic amino acid derivatives differ in curvature, indicating a qualitative difference in the absorption mechanism. An adsorption model for retention is proposed, and arguments are made as to the suitability of an adsorption model for describing the contacts between amino acid side chains during the initial steps of protein folding.     

Formation of Peptide Bonds from Metastable versus Crystalline Phase: Implications for the Origin of Life

Formation of peptide bonds was attempted bythermal activation of dry amino acids from aqueous solutionthat simulated prebiotic evaporative environments. Theevaporation trend of amino acids solutions shows abifurcation and can lead to either a crystalline phase(near equilibrium) or a metastable non-crystalline phase(far from equilibrium). Only amino acids in this metastablephase are able to form peptide bonds by thermal activationat temperatures that are generated by solar radiationtoday. We suggest that this metastable phase is the idealinitial material to trigger amino acid assemblage withprotein-like structure because provide the driving force(supersaturation) for an intense interaction betweenmonomers of different amino acids and allows activation ofthese monomers in plausible prebiotic conditions.     

Analysis on folding of misgurin using two-dimensional HP model

Misgurin is an antimicrobial peptide from the loach, while the hydrophobic-polar (HP) model is a way to study the folding conformations and native states in peptide and protein although several amino acids cannot be classified either hydrophobic or polar. Practically, the HP model requires extremely intensive computations, thus it has yet to be used widely. In this study, we use the two-dimensional HP model to analyze all possible folding conformations and native states of misgurin with conversion of natural amino acids according to the normalized amino acid hydrophobicity index as well as the shortest benchmark HP sequence. The results show that the conversion of misgurin into HP sequence with glycine as hydrophobic amino acid at pH 2 has 1212 folding conformations with the same native state of minimal energy -6; the conversion of glycine as polar amino acid at pH 2 has 13,386 folding conformations with three native states of minimal energy -5; the conversion of glycine as hydrophobic amino acid at pH 7 has 2538 folding conformations with three native states of minimal energy -5; and the conversion of glycine as polar amino acid at pH 7 has 12,852 folding conformations with three native states of minimal energy -4. Those native states can be ranked according to the normalized amino acid hydrophobicity index. The detailed discussions suggest two ways to modify misgurin.     

The effect of the coded amino acids on meso- and ectodermal tissue development in organotypic culture

The effect of L-amino acids was investigated in organotypic tissue culture ofmesodermal tissue (spleen, myocardium) and ectodermal tissue (brain cortex) in mature rats. The low hydrophobic amino acids: asparagine, hystidine, serine, lysine, arginine and glutamine acid, induced the proliferation stimulation. The high hydrophobic amino acids had both the apoptose effect (spleen) and no effect at all (myocardium). The proliferation stimulation occurred in the ectodermal tissue under the effect of the high hydrophobic amino acids (asparagines acid, valine, threonine, methionine, leucine, isoleucine), whereas the low hydrophobic amino acids had no effect on the nervous tissue development. The combination of two amino acids one of which stimulated and another one inhibited the explant growth zone (or was not active in myocardium) lead to an increase of the stimulatory effect in meso- and ectodermal tissue. The amino acid modulated properties can be taken in consideration in synthesis of new regulatory peptides.     

Design of surfactants suitable for protein extraction by reversed micelles

New surfactants have been synthesized for potential use in reversed micellar protein extraction operations. Preferential solubility of the surfactant in an aliphatic solvent such as hexane, heptane, or isooctane and the formation of reversed micelles accompanied with solubilization of significant quantities of water can be achieved by using strongly hydrophobic, twin alkyl chains as the hydrophobic moiety. Different surfactants having identical water-solubilizing capacities can have significantly different behavior in protein extractions, where extraction efficiency appears to be governed by the nature of the interfacial complex that forms between surfactants and proteins. Bulky surfactant chains provide a steric hindrance to the adsorption of the surfactant to the protein surface, thus inhibiting solvation of the protein/surfactant complex, and hence protein extraction. Under these conditions, a precipitate forms either in the bulk aqueous phase or at the interface. Surfactants that can form a close-packed complex with the protein are excellent protein-solubilizing agents. Dioleyl phosphoric acid (DOLPA) appears to be the best surfactant currently available for protein extraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 26-32, 1997.     

A high-throughput competitive scintillation proximity aminoacyl-tRNA synthetase charging assay to measure amino acid concentration

An enhanced method to measure the concentration of individual naturally occurring free amino acids in solution is described. This relatively simple but robust method combines two previously reported procedures: the use of scintillation proximity assay (SPA) technology to measure aminoacyl-tRNA synthetase (aaRS) activity and the use of aaRS activity to measure amino acid concentration using the enzymatic isotope dilution technique. The format described is called an aaRS competitive scintillation proximity assay (cSPA). This cSPA takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same nonradiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid. Under equilibrium conditions, in the case of limiting tRNA, the rate of the enzyme-catalyzed reaction relative to substrate concentration becomes irrelevant and the enzymatic isotopic dilution technique becomes the simple isotopic dilution technique. Due to the exquisite specificity of the reaction, a crude mixture of tRNAs and aaRSs can be used to detect the concentration of a particular amino acid without interference from noncognate amino acids. When used to monitor aminopeptidase M activity, this assay produced similar results in time course and inhibition experiments as compared with a traditional fluorescent assay. High-throughput compatibility was demonstrated by screening 12,000 compounds against aminopeptidase M in 384-well microtiter plates with Z factors ranging from 0.53 to 0.70. This competitive assay can be used as a general method to detect amino acids at concentrations less than 100 nM and to monitor enzyme activity in biological samples, and it is amenable to high-throughput screening.     

44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids.

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the E5 gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the E5 protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the E5 protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.     

A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.