Distal Amino Acid Research Articles

Genetic variants of Helicobacter pylori type IV secretion system components CagL and CagI and their association with clinical outcomes

BackgroundHelicobacter pylori infection is associated with risk for chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). The H. pylori Cag type IV secretion system (TFSS) translocates the virulence factor cytotoxin-associated gene A protein into host cells and plays an important role in initiating gastric carcinogenesis. The CagL and CagI proteins are components of the TFSS. The Arg-Gly-Asp (RGD) motif of CagL, and the six most distal C-terminal amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys) of CagL and CagI are essential for TFSS adhesion to host cells. Additionally, the CagL variant Tyr58Glu59 was previously shown to be associated with GC patients.ResultsWe isolated 43 H. pylori isolates from 17 CG, 8 GU, 8 DU, and 10 GC patients in Southeast Asia. Total DNAs were extracted and sequenced with MiSeq. H. pylori strain ATCC 26695, which was isolated from CG patients, was used as a reference. We examined the full sequences of H. pylori cagL and cagI using whole-genome sequencing (WGS), and analyzed whether single nucleotide variants and amino acid changes (AACs) correlated with adverse clinical outcomes. Three isolates were excluded from the analysis due to cagPAI rearrangements. CagL RGD motifs were conserved in 39 isolates (97.5%). CagL-Glu59 and Ile234 in the C-terminal motif were more common in 10 H. pylori isolates from GC patients (p < 0.001 and p < 0.05, respectively). When 5 Vietnamese isolates from GC patients were excluded, CagL-Glu59 still remains significant (p < 0.05), but not Ile234. CagL-Tyr58 was seen in only one isolate. The CagI C-terminal motif was completely conserved across all 40 isolates, and there were no significant AACs in CagI.ConclusionsUsing WGS, we analyzed genetic variants in clinical H. pylori isolates and identified putative novel and candidate variants in uncharacterized CagL and CagI sequences that are related to gastric carcinogenesis. In particular, CagL-Glu59 has the possible association with GC.

Extended Electron-Transfer in Animal Cryptochromes Mediated by a Tetrad of Aromatic Amino Acids

The cryptochrome/photolyase protein family possesses a conserved triad of tryptophans that may act as a molecular wire to transport electrons from the protein surface to the FAD cofactor for activation and/or signaling-state formation. Members from the animal (and animal-like) cryptochrome subclade use this process in a light-induced fashion in a number of exciting responses, such as the (re-)setting of circadian rhythms or magnetoreception; however, electron-transfer pathways have not been explored in detail yet. Therefore, we present an in-depth time-resolved optical and electron-paramagnetic resonance spectroscopic study of two cryptochromes from Chlamydomonas reinhardtii and Drosophila melanogaster. The results do not only reveal the existence of a fourth, more distant aromatic amino acid that serves as a terminal electron donor in both proteins, but also show that a tyrosine is able to fulfill this very role in Chlamydomonas reinhardtii cryptochrome. Additionally, exchange of the respective fourth aromatic amino acid to redox-inactive phenylalanines still leads to light-induced radical pair formation; however, the lifetimes of these species are drastically reduced from the ms- to the μs-range. The results presented in this study open up a new chapter, to our knowledge, in the diversity of electron-transfer pathways in cryptochromes. Moreover, they could explain unique functions of animal cryptochromes, in particular their potential roles in magnetoreception because magnetic-field effects of light-induced radical pairs strongly depend on distance and orientation parameters.

Reciprocal voltage sensor-to-pore coupling leads to potassium channel C-type inactivation.

Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions – a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd2+ bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K+ coordination, a hallmark for C-type inactivation. An engineered Cd2+ bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

PARTICIPATION OF Y89 AND Y97 IN THE CONJUGATING ACTIVITY OF Drosophila melanogaster GLUTATHIONE S-TRANSFERASE D3 (DmGSTD3).

Drosophila melanogaster glutathione S-transferase D3 (DmGSTD3) has a shorter amino acid sequence as compared to other GSTs known in the fruit flies. This is due to the 15 amino acid N-terminal truncation in which normally active amino acid residue is located. The work has made use of homology modeling to visualize the arrangement of amino acid side chains in the glutathione (GSH) substrate cavity. The identified amino acids were then replaced with amino acids without functional groups in the side chains and the mutants were analyzed kinetically. Homology modeling revealed that the side chains of Y89 and Y97 were shown facing toward the substrate cavity proposing their possible role in catalyzing the conjugation. Y97A and Y89A GSH gave large changes in Km (twofold increase), Vmax (fivefold reduction), and Kcat /Km values for GSH suggesting their significant role in the conjugation reaction. The replacement at either positions has not affected the affinity of the enzyme toward 1-chloro-2,4-dinitrobenzene as no significant change in values of Kmax was observed. The replacement, however, had significantly reduced the catalytic efficiency of both mutants with (Kcat /Km )(GSH) and (Kcat /Km )(CDNB) of eight- and twofold reduction. The recombinant DmGSTD3 has shown no activity toward 1,2-dichloro-4-nitrobenzene, 2,4-hexadienal, 2,4-heptadienal, p-nitrobenzyl chloride, ethacrynic acid, and sulfobromophthalein. Therefore, it was evident that DmGSTD3 has made use of distal amino acids Y97 and Y89 for GSH conjugation.

Dissecting the roles of local packing density and longer-range effects in protein sequence evolution.

What are the structural determinants of protein sequence evolution? A number of site-specific structural characteristics have been proposed, most of which are broadly related to either the density of contacts or the solvent accessibility of individual residues. Most importantly, there has been disagreement in the literature over the relative importance of solvent accessibility and local packing density for explaining site-specific sequence variability in proteins. We show that this discussion has been confounded by the definition of local packing density. The most commonly used measures of local packing, such as contact number and the weighted contact number, represent the combined effects of local packing density and longer-range effects. As an alternative, we propose a truly local measure of packing density around a single residue, based on the Voronoi cell volume. We show that the Voronoi cell volume, when calculated relative to the geometric center of amino-acid side chains, behaves nearly identically to the relative solvent accessibility, and each individually can explain, on average, approximately 34% of the site-specific variation in evolutionary rate in a data set of 209 enzymes. An additional 10% of variation can be explained by nonlocal effects that are captured in the weighted contact number. Consequently, evolutionary variation at a site is determined by the combined effects of the immediate amino-acid neighbors of that site and effects mediated by more distant amino acids. We conclude that instead of contrasting solvent accessibility and local packing density, future research should emphasize on the relative importance of immediate contacts and longer-range effects on evolutionary variation. Proteins 2016; 84:841-854. © 2016 Wiley Periodicals, Inc.

A Reciprocal Voltage Sensor-To-Pore Coupling in C-Type Inactivation

Voltage-gated potassium channels undergo slow (C-type) inactivation upon persistent voltage stimulation; channel opening leads to a molecular rearrangement of the selectivity filter, shutting down potassium conduction. An important question is how the peripherally located voltage-sensor domain (VSD) can affect the inactivation of the pore domain, and conversely, how the pore domain can affect the VSD with respect to C-type inactivation. We studied the Shaker K+ channel expressed in Xenopus oocytes by the two-electrode voltage clamp technique. We showed experimentally that pulling residue 416 in the pore domain towards the VSD by a Cd2+ bridge accelerates C-type inactivation. In line with this, molecular dynamics simulations show that such pulling widens the selectivity filter, a hallmark for C-type inactivation. Experimentally induced rotations of the voltage sensor S4 and an engineered Cd2+ bridge within the VSD also affects C-type inactivation; conversely, a pore domain mutation affects VSD gating charge movement. Finally, experimental data suggests that C-type inactivation is caused by a concerted action of distant amino acid residues in the pore domain. These data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.

Proximal Pocket Hydrogen Bonds Significantly Influence the Mechanism of Chloroperoxidase Compound I Formation.

The influence of backbone hydrogen bonds to the sulfur atom of the proximal thiolate (NH···S hydrogen bonds) on the formation of compound I in chloroperoxidase is investigated with DFT calculations. Reaction profiles for the transformation of the ferric resting state into compound I in the presence of a peroxide substrate are calculated for a model system incorporating the heme and key proximal and distal amino acid residues. We find that NH···S hydrogen bonds (1) reduce the barrier for the formation of compound 0 by 7.6 kcal/mol, (2) increase the stability of compound 0 by 5.2 kcal/mol, (3) reduce the stability of compound I relative to compound 0 by 6.2 kcal/mol, and (4) reduce the stability of protonated compound 0, favoring a hybrid homo-heterolytic relative to a classic heterolytic mechanism for O-O bond scission. In general, the influence of the NH···S hydrogen bonds can be traced to a reduction in the pKa of the heme-bound substrate. We find that the hydrogen bond networks on the proximal and distal sides of the heme function together to modulate the mechanism of reaction. These results confirm and extend long-standing theories that the NH···S hydrogen bonds in heme thiolate proteins influence reactivity by tuning the thiolate "push" effect.

Serologic epitopes defined across multiple exons

Aim Identification of anti-HLA antibody specificities with complex molecular definition across multiple exons. Methods Epitope analysis of anti-HLA antibody specificity studies using Luminex single-antigen beads. Results B∗42:01:01 has the same exon 2 as B∗07:02:01 and the same exon 3 as B∗08:01:01. B∗42:01:01 should then share with B∗07:02:01 the epitopes coded by exon 2 and with B∗08:01:01 the epitopes coded by exon 3. In fact, antibody reactivity patterns show that sera reactive with B∗42:01:01 is typically also reactive with B∗08:01:01 or B∗07:02:01 or both. Nevertheless there are sera that is reactive with B∗42:01:01 but not with either B∗08:01:01 or B∗07:02:01. One might think that this unique reactivity must be against an epitope in a section of the protein in the transition of the segment coded by exon 2 and that coded by exon 3, which happens to be amino acid position 91, absent in both B∗07:02:01 and B∗08:01:01. But these two alleles have the same sequence in the 72–113 amino acid position segment, so the epitope that explains this reactivity must be outside this segment. A possible definition of this epitope can be provided by isoform 67(Y)-69(A)-70(Q)-71(A) in the first alpha helix and 114(N) in the second beta-pleated sheet, in relatively close spatial proximity. The isoform present in B∗42:01:01 for this epitope is not present in either B∗08:01:01 or B∗07:02:01. It is, however, present in B∗54:01:01, B∗55:01:01, B∗55:02:01, B∗56:01:01, B∗81:01 and B∗82:01. Out of 4 serum samples with reactivity against B∗42:01:01, but not against either B∗07:02:01 or B∗08:01:01, 3 of them had also reactivity against B∗54:01:01, B∗55:01:01, B∗55:02:01, B∗56:01:01, B∗81:01 and B∗82:01. Other examples exists such as variations of the Bw4 where B∗13:01:01 is excluded, defined not only positions 82-83 coded by exon 2, but also by position 145 coded by exon 3. Conclusions The molecular definition of serologic epitopes is often simple in the form of close amino acid positions, but on other occasions it requires the complex interaction of distant amino acid positions. Algorithmic predictions of serologic epitopes is unsuccessful in the identification of these complex epitopes, which can only be identified by careful analysis of empirical data.

Interplay of the H-bond donor-acceptor role of the distal residues in hydroxyl ligand stabilization of Thermobifida fusca truncated hemoglobin.

The unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH(-) group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe-OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family.

“Backdoor Induction” of Chirality: Asymmetric Hydrogenation with Rhodium(I) Complexes of Triphenylphosphane-Substituted β-Turn Mimetics

Bioconjugate bidentate ligands 2–10 were obtained by tethering triphenylphosphanecarboxylic acid to amino acid substituted spacers with different flexibility, ranging from a rigid enediyne-based β-turn inducer to flexible linear aliphatic chains with up to eight carbon atoms. The 21 synthesized ligands revealed up to 81% ee selectivity in rhodium-catalyzed asymmetric hydrogenation of α,β-unsaturated amino acids. The key feature of the catalysts is the prochiral coordination sphere of the catalytic metal while the chirality is transmitted by “backdoor induction” from distant hydrogen-bonded amino acids. DFT calculations were applied to study the structure and relative stability of the precatalytic organometallic Rh(I) complexes, with particular emphasis on hydrogen-bonded secondary structures.

ChemInform Abstract: “Backdoor Induction” of Chirality in Asymmetric Hydrogenation with Rhodium(I) Complexes of Amino Acid Substituted Triphenylphosphane Ligands.

This paper describes the synthesis and characterization of 5-(di-phenylphosphino)isophthalic acid bioconjugates, Lig-[R]2. In addition to symmetrically disubstituted conjugates with amino acids (1a-1e and 4f-4g), peptides (2a-2e and 3a-3b) or amines (4a-4e), a convenient one pot two step procedure for the synthesis of conjugates 5a-g bearing two different substituents is reported. The 28 prepared phosphanes 1-5 are used as monodentate ligands in the rhodium(I) catalyzed hydrogenation of 2-acetamidoacrylate or (Z)-(alpha)-acetamidocinnamate. Ligand with small side chain substituents Lig-[Ala-OMe]2 1a revealed highest selectivity with up to 84 % ee. The catalysts presented herein are models of artificial metalloenzymes where the secondary coordination sphere controls the selectivity in catalysis. The chirality of distant hydrogen-bonded amino acids is transmitted by backdoor induction to the prochiral Rh(I).

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.

Small ligand–globin interactions: Reviewing lessons derived from computer simulation

In this work we review the application of classical and quantum-mechanical atomistic computer simulation tools to the investigation of small ligand interaction with globins. In the first part, studies of ligand migration, with its connection to kinetic association rate constants (kon), are presented. In the second part, we review studies for a variety of ligands such as O2, NO, CO, HS−, F−, and NO2− showing how the heme structure, proximal effects, and the interactions with the distal amino acids can modulate proteinligand binding. The review presents mainly results derived from our previous works on the subject, in the context of other theoretical and experimental studies performed by others. The variety and extent of the presented data yield a clear example of how computer simulation tools have, in the last decade, contributed to our deeper understanding of small ligand interactions with globins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. 1H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00ppm, GlnE7Nε1H/Nε2H at 10.66ppm/−3.27ppm, and PheE11 CδH at 11.75ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen bonding network loop between the 6-propionate, the heme ligand and nearby amino acids, tailoring in this way the electron density in the heme-ligand moiety.

The application of “backdoor induction” in bioinspired asymmetric catalysis

Amino acid substituted monodentate triphenylphosphine ligands Lig-Aa1-Aa2-Aa3-Z 5 and 6, with a C-terminal ester or amide group, respectively, were prepared in a few simple synthetic steps. Supramolecular in situ formed complexes [Rh(COD)(Lig-Aa1-Aa2-Aa3-Z)2]BF43 and 4 with a prochiral coordination sphere of the metal are selective hydrogenation catalysts (up to 68% ee). The selectivity is induced by transmission of chirality via distant hydrogen-bonded amino acids with a Herrick-like secondary structure (“backdoor induction”).

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600–620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450–460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin ( Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.

Influence of sequence variability on bactericidal activity sera induced by Factor H binding protein variant 1.1

Factor H binding protein (fHbp), one of the main antigens of new vaccines against serogroup B meningococcus, varies in amino acid sequence and level of expression in different clinical isolates. To evaluate the contribution of amino acid sequence variability to vaccine coverage, we constructed a strain that is susceptible to bactericidal killing only by anti-fHbp antibodies and engineered it to express equal levels of 10 different fHbp sub-variants from a constitutive promoter. Testing of these isogenic strains showed that sera from mice or adult volunteers vaccinated with fHbp variant 1.1 were bactericidal against all sub-variants 1 sequences, however the titer against the most distant sequences were several times lower. Sera from vaccinated infants were more susceptible to amino acid variations and they had lower or no bactericidal activity against the distant sub-variants 1 sequences in comparison with sera from adults given the same vaccines. The low coverage provided by fHbp could be overcome using a multicomponent vaccine. We conclude that fHbp is a very important antigen that induces bactericidal antibodies in animals, adults and infants. However, given its high variability of sequence and expression level, it is unlikely that fHbp alone can provide good protection in infants against the distant amino acid sequence variants and therefore multicomponent vaccines inducing protective immunity also against other antigens are more likely to induce a broad protective immunity in all age groups.

A metagenomic β-glucuronidase uncovers a core adaptive function of the human intestinal microbiome.

In the human gastrointestinal tract, bacterial β-D-glucuronidases (BG; E.C. 3.2.1.31) are involved both in xenobiotic metabolism and in some of the beneficial effects of dietary compounds. Despite their biological significance, investigations are hampered by the fact that only a few BGs have so far been studied. A functional metagenomic approach was therefore performed on intestinal metagenomic libraries using chromogenic glucuronides as probes. Using this strategy, 19 positive metagenomic clones were identified but only one exhibited strong β-D-glucuronidase activity when subcloned into an expression vector. The cloned gene encoded a β-D-glucuronidase (called H11G11-BG) that had distant amino acid sequence homologies and an additional C terminus domain compared with known β-D-glucuronidases. Fifteen homologs were identified in public bacterial genome databases (38-57% identity with H11G11-BG) in the Firmicutes phylum. The genomes identified derived from strains from Ruminococcaceae, Lachnospiraceae, and Clostridiaceae. The genetic context diversity, with closely related symporters and gene duplication, argued for functional diversity and contribution to adaptive mechanisms. In contrast to the previously known β-D-glucuronidases, this previously undescribed type was present in the published microbiome of each healthy adult/child investigated (n = 11) and was specific to the human gut ecosystem. In conclusion, our functional metagenomic approach revealed a class of BGs that may be part of a functional core specifically evolved to adapt to the human gut environment with major health implications. We propose consensus motifs for this unique Firmicutes β-D-glucuronidase subfamily and for the glycosyl hydrolase family 2.

Molecular Dynamics and Docking Studies of Single Site Esterase Peptide Dendrimers

Herein we report the first molecular dynamics and docking study of peptide dendrimers, at the example of dendrimers catalyzing the hydrolysis of acyloxy pyrene trisulfonates in aqueous buffer. Molecular dynamics provide models comparable to the observation by diffusion-NMR showing that the dendrimers exist as conformationally flexible molten globules in aqueous solution. Packing is evidenced by the occurrence of close contacts between topologically distant amino acids in the dendrimer model. Backbone hydrogen bonds predominantly form 2, 3, or 4 residues apart as found in the secondary structures of proteins, however, with lower frequency. The catalytic residues are present at the surface of the dendrimer model at relative positions compatible with binding and esterolysis. Docking of the substrate to low-energy conformations of the dendrimers predicts the formation of dendrimer-substrate complexes with one or two salt bridges between the sulfonate and protonated arginine or histidine residues. Substrate binding in the docked models also involves 4-6 van der Waals contacts. In the catalytic dendrimers RG3 and RMG3 exhibiting a positive dendritic effect, these docking contacts involve the outer dendritic branches. By contrast, in the catalytic dendrimers HG3 or HMG3 where no such effect occurs, the docking contacts are concentrated at the dendritic core. The present investigations provide an unprecedented insight into the molecular dynamics of peptide dendrimers that may be of general significance for other conformationally flexible dendrimers.

Paramagnetic 13C and 15N NMR Analyses of Cyanide- (13C15N-) Ligated Ferric Peroxidases: The Push Effect, Not Pull Effect, Modulates the Compound I Formation Rate

Paramagnetic (13)C and (15)N NMR spectroscopy of heme-bound cyanide ((13)C(15)N) was utilized to quantitatively distinguish the electron donor effect (the push effect) from the proximal histidine and hydrogen-bonding effect (the pull effect) from the distal amino acid residues in cytochrome c peroxidase (CcP), ascorbate peroxidase (APX), lignin peroxidase (LiP), and manganese peroxidase (MnP). Paramagnetic (13)C NMR signals of heme-bound (13)C(15)N of these peroxidases were observed in a wide range, -3501 ppm (CcP), -3563 ppm (APX), -3823 ppm (MnP), and -3826 ppm (LiP), while paramagnetic (15)N NMR signals of those were detected in a narrow range, 574 ppm (ARP), 605 ppm (CcP), 626 ppm (LiP), and 654 ppm (MnP). Detailed analysis, combined with the previous results for horseradish peroxidase and Arthromyces ramosus peroxidase, indicated that the push effect is quite different among these peroxidases while the pull effect is similar. More importantly, a strong correlation between the (13)C NMR shift (the push effect) and the compound I formation rate was observed, indicating that the push effect causes a variation in the compound I formation rate. Comparison of the (13)C and (15)N NMR results of these peroxidases with their crystal structures suggests that the orientation of the proximal imidazole plane to the heme N-Fe-N axis controls the push effect and the compound I formation rate of peroxidase.