Use of homoarginine to obtain attenuated cationic membrane lytic peptides.

Our research group has been studying the design of intracellular delivery peptides based on cationic lytic peptides. By placing negatively charged amino acids on potentially hydrophobic faces of the peptides, membrane lytic activity is attenuated on the cell surface, whereas it recovers in endosomes, enabling cytosolic delivery of proteins including antibodies. These lytic peptides generally contain multiple lysines, facilitating cell surface interaction and membrane perturbation. This study evaluated the effect of lysine-to-homoarginine substitution using HAad as a model delivery peptide. The resulting peptide had a comparable or better delivery efficacy for Cre recombinase, antibodies, and the Cas9/sgRNA complex with one-quarter of the concentration of HAad, implying that a subtle structural difference can affect delivery activity.

Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of α-Heteroatom Assistance.

Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (2H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors 1H or 2H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors 2H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.

Study of the affinity between the protein kinase PKA and homoarginine-containing peptides derived from kemptide: Free energy perturbation (FEP) calculations.

Protein kinases (PKs) discriminate between closely related sequences that contain serine, threonine, and/or tyrosine residues. Such specificity is defined by the amino acid sequence surrounding the phosphorylatable residue, so that it is possible to identify an optimal recognition motif (ORM) for each PK. The ORM for the protein kinase A (PKA), a well-known member of the PK family, is the sequence RRX(S/T)X, where arginines at the -3 and -2 positions play a key role with respect to the primed phosphorylation site. In this work, differential affinities of PKA for the peptide substrate Kemptide (LRRASLG) and mutants that substitute the arginine residues by the unnatural peptide homoarginine were evaluated through molecular dynamics (MD) and free energy perturbation (FEP) calculations. The FEP study for the homoarginine mutants required previous elaboration of a CHARMM "arginine to homoarginine" (R2B) hybrid topology file which is available in this manuscript as Supporting Information. Mutants substituting the arginine residues by alanine, lysine, and histidine were also considered in the comparison by using the same protocol. FEP calculations allowed estimating the free energy changes from the free PKA to PKA-substrate complex (ΔΔGE→ES ) when Kemptide structure was mutated. Both ΔΔGS→ES values for homoarginine mutants were predicted with a difference below 1 kcal/mol. In addition, FEP correctly predicted that all the studied mutations decrease the catalytic efficiency of Kemptide for PKA. © 2018 Wiley Periodicals, Inc.

O-Methylisourea Can React with the α-Amino Group of Lysine: Implications for the Analysis of Reactive Lysine.

The specificity of O-methylisourea (OMIU) to bind to the ε-amino group of Lys, an important supposition for the OMIU-reactive Lys analysis of foods, feeds, ingredients, and digesta, was investigated. Crystalline l-Lys incubated under standard conditions with OMIU resulted in low homoarginine recoveries. The reaction of OMIU with the α-amino group of Lys was confirmed by MS analysis, with double derivatized Lys being identified. None of the changes in reaction conditions (OMIU pH, OMIU to Lys ratio, and reaction time) with crystalline l-Lys resulted in 100% recovery of homoarginine. The average free Lys content in ileal digesta of growing pigs and broilers was found to be 13% of total Lys, which could result in a significant underestimation of the reactive Lys content. The reaction of OMIU with α-amino groups may necessitate analysis of free Lys to accurately quantify reactive lysine in samples containing a large proportion of Lys with a free α-amino group.

Simultaneous stable-isotope dilution GC-MS measurement of homoarginine, guanidinoacetate and their common precursor arginine in plasma and their interrelationships in healthy and diseased humans.

Low concentrations of L-homoarginine (hArg) in plasma or serum and urine have recently emerged as a novel cardiovascular risk factor. Previously, we reported gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS (GC-MS/MS) methods for the quantitative determination of hArg and Arg in plasma, serum, urine and other biological samples. In these methods, plasma and serum are ultrafiltered by means of commercially available cartridges (10 kDa), and 10-µL ultrafiltrate aliquots are subjected to a two-step derivatization procedure, yielding the methyl ester tri(N-pentafluoropropionyl) derivatives. De novo prepared trideuteromethyl ester hArg (d3Me-hArg) was used as an internal standard. To make the hArg analysis in plasma more convenient, straightforward and cheaper we performed two key modifications: (1) precipitation of plasma proteins by methanol and (2) use of newly prepared and d3Me-hArg as the internal standard. The method was validated and used for the quantitative determination of hArg in human plasma by GC-MS after electron-capture negative-ion chemical ionization (ECNICI) using methane as the reactant gas. Intra-assay accuracy (recovery) and imprecision (relative standard deviation) were within generally accepted ranges (93-109 and 2.3-10 %, respectively). Furthermore, we extended the applicability of this method to guanidinoacetate (GAA). This is of particular importance because hArg and GAA are produced from Arg by the catalytic action of arginine:glycine amidinotransferase (AGAT) also known as glycine:arginine transamidinase (GATM). Using this method, we quantitated simultaneously hArg, Arg and GAA in the selected-ion monitoring mode in 10-µL aliquots of plasma. In plasma samples of 17 non-medicated healthy young men, the concentration of hArg, GAA and Arg was determined to be (mean ± SD) 1.7 ± 0.6, 2.6 ± 0.8, 91 ± 29 µM, respectively. The correlation between hArg and Arg was borderline (r = 0.47, P = 0.06). GAA strongly correlated with Arg (r = 0.82, P < 0.0001) but did not correlate with hArg (r = 0.17, P = 0.52). The plasma concentrations of hArg, GAA and Arg measured in 9 patients suffering from stroke or transitory ischemic attack were 1.8 ± 0.6, 2.7 ± 0.4 and 82 ± 17 µM. The ratio values of the hArg, GAA and Arg concentrations measured after removal of plasma proteins by methanol precipitation or ultrafiltration were 0.94 ± 0.1, 0.94 ± 0.08, and 0.88 ± 0.07, respectively. Simultaneous measurement of hArg and GAA in human plasma may allow assessment of AGAT activity in vivo with respect both to GAA and to hArg and their relationship in health, disease, nutrition and pharmacotherapy.

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli.

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to L-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize L-homoarginine and L-N(6)-(1-iminoethyl)lysine (L-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA(Pyl) CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host's ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to L-homoarginine or L-NIL. The assignment of AGG to L-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.

Influence of L-homoarginine as an analogue of L-arginine on the heat-induced aggregation of proteins.

The influence of l-homoarginine on the heat-induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l-homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l-Homoarginine increased the onset temperature of heat-induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l-homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l-homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur.

Solid-phase N-terminal peptide enrichment study by optimizing trypsin proteolysis on homoarginine-modified proteins by mass spectrometry.

RATIONALE: Proteolytic cleavages generate active precursor proteins by creating new N-termini in the proteins. A number of strategies have recently been published regarding the enrichment of original or newly formed N-terminal peptides using guanidination of lysine residues and amine-reactive reagents. For effective enrichment of N-terminal peptides, the efficiency of trypsin proteolysis on homoarginine (guanidinated) modified proteins must be understood and simple and versatile solid-phase N-terminal capture strategies should be developed. METHODS: We present here a mass spectrometry (MS)-based study to evaluate and optimize the trypsin proteolysis on a guanidinated-modified protein. Trypsin proteolysis was studied using different amounts of trypsin to modified protein ratios. To capture the original N-termini, after guanidination of proteins, original N-termini were acetylated and the proteins were digested with trypsin. The newly formed N-terminal tryptic peptides were captured with a new amine reactive acid-cleavable solid-phase reagent. The original N-terminal peptides were then collected from the supernatant of the solution. RESULTS: We demonstrated a detailed study of the efficiency of enzyme trypsin on homoarginine-modified proteins. We observed that the rate of hydrolysis of homoarginine residues compared to their lysine/arginine counterparts were slower but generally cleaved after an overnight digestion period depending on the protein to protease concentration ratios. Selectivity of the solid-phase N-terminal reagent was studied by enrichment of original N-terminal peptides from two standard proteins, ubiquitin and RNaseS. CONCLUSIONS: We found enzyme trypsin is active in the guanidinated form of the protein depending on the enzyme to protein concentrations, time and the proximity of arginine residues in the sequence. The novel solid-phase capture reagent also successfully enriched N-terminal peptides from the standard protein mixtures. We believe this trypsin proteolysis study on homoarginine-modified proteins and our simple and versatile solid-phase capture strategy could be very useful for enrichment and sequence determination of proteins N-termini by MS.

Determination of homoarginine, arginine, NMMA, ADMA, and SDMA in biological samples by HPLC-ESI-mass spectrometry.

N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G)-methyl-L-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, N(G),N'(G)-dimethyl-L-arginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with L-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. L-homoarginine (HArg), a structural analog of L-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of L-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.

Interactions between L-arginine/L-arginine derivatives and lysozyme and implications to their inhibition effects on protein aggregation.

L-arginine (Arg), L-homoarginine (HArg), L-arginine ethylester (ArgEE), and L-arginine methylester (ArgME) were found effective in inhibiting protein aggregation, but the molecular mechanisms are not clear. Herein, stopped-flow fluorescence spectroscopy, isothermal titration calorimetry, and mass spectroscopy were used to investigate the folding kinetics of lysozyme and the interactions of the additives with lysozyme. It was found that the interactions of ArgME and ArgEE with lysozyme were similar to that of guanidine hydrochloride and were much stronger than those of Arg and HArg. The binding forces were all mainly hydrogen bonding and cation-π interaction from the guanidinium group, but their differences in molecular states led to the significantly different binding strengths. The additives formed molecular clusters in an increasing order of ArgEE, ArgME, HArg, and Arg. Arg and HArg mainly formed annular clusters with head-to-tail bonding, while ArgME and ArgEE formed linear clusters with guanidinium groups stacking. The interactions between the additives and lysozyme were positively related to the monomer contents. That is, the monomers were the primary species that participated in the direct interactions due to their intact guanidinium groups and small sizes, while the clusters performed as barriers to crowd out the protein-protein interactions for aggregation. Thus, it is concluded that the effects of Arg and its derivatives on protein aggregation stemmed from the direct interactions by the monomers and the crowding effects by the clusters. Interplay of the two effects led to the differences in their inhibition effects on protein aggregation.

Novel apidaecin 1b analogs with superior serum stabilities for treatment of infections by gram-negative pathogens.

Proline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with l-ornithine or l-homoarginine increased the serum stabilities by more than 20-fold (half-life, ∼4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N,N,N',N'-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and d-amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.

Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase.

Arginine analogs were incorporated site-specifically into proteins using an in vitro translation system. In this system, mRNAs containing a CGGG codon were translated by an aminoacyl-tRNA(CCCG), which was charged with arginine analogs using yeast arginyl-tRNA synthetase. N(G)-monomethyl-L-arginine, L-citrulline and L-homoarginine were incorporated successfully into proteins using this method. The influence of arginine monomethylation in histone H3 on the acetylation of lysine residues by histone acetyltransferase hGCN5 was investigated, and the results demonstrated that K9 acetylation was suppressed by the methylation of R8 and R17 but not by R26 methylation. K18 acetylation was not affected by the methylation of R8, R17 and R26. This site-specific modification strategy provides a way to explore the roles of post-translational modifications in the absence of heterogeneity due to other modifications.

Role of the guanidine group in the N-terminal fragment of PTH(1-11).

A series of PTH hybrids containing a diamine [NH(2)(CH(2))(n)NH(2); n = 4, 5, 6] in the C-terminal position was synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH(2) (Har = homoarginine) template. The compounds were pharmacologically characterized at PTH1R receptors for agonist activity.

Chemical modulation of the chaperone function of human alphaA-crystallin.

alphaA-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino group of selected arginine residues by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), makes human alphaA-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of alphaA-crystallin. alphaA-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by approximately 40% and approximately 60% beyond that of unmodified alphaA-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU treatment reduced the surface hydrophobicity but after MGO treatment, it was approximately 39% higher than control. FRET analysis revealed that alphaA-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of alphaA-crystallin in check and that a metabolic alpha-dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.

Synthesis and biological activity of homoarginine-containing opioid peptides.

Two tris-alkoxycarbonyl homoarginine derivatives, Boc-Har{omega,omega'-[Z(2Br)]2}-OH and Boc-Har{omega,omega'-[Z(2Cl)]2}-OH, were prepared by guanidinylation of Boc-Lys-OH, and used for the synthesis of neo-endorphins and dynorphins. The results were compared with that obtained in the synthesis in which Boc-Lys(Fmoc)-OH was incorporated into the peptide chain, and after removing Fmoc protection, the resulting peptide-resin was guanidinylated with N,N'-[Z(2Br)]2- or N,N'-[Z(2Cl)]2-S-methylisourea. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. The results indicated that replacement of Arg by Har may be a good avenue for the design of biologically active peptides with increased resistance to degradation by trypsin-like enzymes.

Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted alpha-amylase from Pseudoalteramonas haloplanktis.

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis (AHA) is a multidomain enzyme capable of reversible unfolding. Cold-adapted proteins, including AHA, have been predicted to be structurally flexible and conformationally unstable as a consequence of a high lysine-to-arginine ratio. In order to examine the role of low arginine content in structural flexibility of AHA, the amino groups of lysine were guanidinated to form homo-arginine (hR), and the structure-function-stability properties of the modified enzyme were analyzed by transverse urea gradient-gel electrophoresis. The extent of modification was monitored by MALDI-TOF-MS, and correlated to changes in activity and stability. Modifying lysine to hR produced a conformationally more stable and less active alpha-amylase. The k(cat) of the modified enzyme decreased with a concomitant increase in deltaH# and decrease in K(m). To interpret the structural basis of the kinetic and thermodynamic properties, the hR residues were modeled in the AHA X-ray structure and compared to the X-ray structure of a thermostable homolog. The experimental properties of the modified AHA were consistent with K106hR forming an intra-Domain B salt bridge to stabilize the active site and decrease the cooperativity of unfolding. Homo-Arg modification also appeared to alter Ca2+ and Cl- binding in the active site. Our results indicate that replacing lysine with hR generates mesophilic-like characteristics in AHA, and provides support for the importance of lysine residues in promoting enzyme cold adaptation. These data were consistent with computational analyses that show that AHA possesses a compositional bias that favors decreased conformational stability and increased flexibility.

Chemical and biological investigations of beta-oligoarginines.

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the 'protein transduction' by beta-oligoarginines are discussed.

Computational docking of L-arginine and its structural analogues to C-terminal domain of Escherichia coli arginine repressor protein (ArgRc).

The arginine repressor (ArgR) of Escherichia coli binds to six L-arginine molecules that act as its co-repressor in order to bind to DNA. The binding of L-arginine molecules as well as its structural analogues is compared by means of computational docking. A grid-based energy evaluation method combined with a Monte Carlo simulated annealing process was used in the automated docking. For all ligands, the docking procedure proposed more than one binding site in the C-terminal domain of ArgR (ArgRc). Interaction patterns of ArgRc with L-arginine were also observed for L-canavanine and L-citrulline. L-lysine and L-homoarginine, on the other hand, were shown to bind poorly at the binding site. Figure A general overview of the sites found from docking the various ligands into ArgRc ( grey ribbons). Red coloured sticks: residues in binding site H that was selected for docking

Oriented versus random protein immobilization.

Immobilization of proteins on solid surfaces plays an important role in all the fields of modern biology. Two approaches are used in the immobilization of proteins: a random and an oriented mode of binding to solid matrices. In this note, we show that there is not much difference in using either mode of immobilization, since proteins usually bind to a matrix through only one or two bonds. This is demonstrated by the attachment of several proteins to CNBr-activated Sepharose through their lysines and the consequent conversion of those lysines to homoarginine upon treatment with ammonium hydroxide.