Tryptophan In Position Research Articles

Photoredox C(2)-Arylation of Indole- and Tryptophan-Containing Biomolecules.

We introduce a novel and straightforward methodology for photoredox arylation of an indole scaffold using aryldiazonium salts under mild and metal-free conditions. Our approach enables the regioselective and chemoselective introduction of several aryl groups to the C(2) position of indoles and tryptophan, even in competition with other amino acids. This approach extends to the late-stage functionalization of peptides and lysozyme, heralding the unprecedented arylation of tryptophan residues in wild-type proteins and offering broad utility in chemical biology.

Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

Analysis of role of aromatic residues in extracellular loop 2 of Prokineticin receptor 2 in ligand binding probed with genetically encoded photo-crosslinkers

Prokineticin 2 (PK2) and Prokineticin 2 beta (PK2β), products of alternative splicing of pk2 gene, are chemokine-like proteins. While PK2 mediates its biological activities by signaling with the same efficiency through two homologous G protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2), PK2β is able to bind specifically PKR1.Extracellular loop 2 (ECL2) of chemokine receptors is a part of a transmembrane (TM) ligand binding site. In the ECL2 of PKR2 is present, as well as in all chemokine receptors, an aromatic residue cluster, involving tryptophan 212 localized four residues after an ECL2 conserved cysteine, and Phenylalanine 198 located in the top of TM 4.In this work, the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine is incorporated by amber codon suppression technology into PKR2 in position 212. Experiments of photoactivatable cross-linking demonstrated the role of tryptophan in position 212 for binding the ligand contacting Tryptophan in position 24. We also analyzed the role of Phenylalanine 198 in the specificity of PKRs binding. The comparison of TM-bundle binding sites between PKR1 and PKR2 revealed that they are completely conserved except for one residue: valine 207 in human PKR1, which is phenylalanine 198 in human PKR2. The F198V mutation in PKR2 permits to obtain a receptor able to bind more efficiently PK2β, a ligand highly specific for PKR1.

C4 Pictet–Spengler Reactions for the Synthesis of Core Structures in Hyrtiazepine Alkaloids

The hyrtiazepine alkaloids are a family of bisindole natural products that have the azepinoindole backbone. We developed a biomimetic approach by constructing the azepinoindole core in a one-pot manner through 1,4-diazabicyclo[2.2.2]octane/2,2,2-trifluoroethanol (DABCO/TFE) promoted Pictet–Spengler reaction onto the C-4 position of tryptophan. This strategy allowed the synthesis of common key structures of these families. The key intermediate can be converted into the 3H-pyrano[2,3-b:5,6-e′]diindol intermediate present in hyrtimomines A and B, as well as the azepinoindole core present in fargesine.

Membrane Structural Analysis by Enhanced Raman Scattering

Gold nanoparticles focus light to a molecular length scale, creating the possibility to visualize molecular structure. The high optical intensity at a nanostructure surface leads to surface enhanced Raman scattering (SERS) from molecules nearby. Due to the surface alignment and rapid decay of the electromagnetic near field, SERS spectra contain information on molecular position and orientation relative to the surface, but they are difficult to interpret quantitatively. Here we report a ratiometric analysis that combines SERS and unenhanced Raman spectra with polarizability tensors calculated by time dependent density functional theory (TDDFT), and electromagnetic (EM) near fields from numerical simulations. The analysis was applied to gold nanorods surrounded by fluid phase dioleoylphosphatidylcholine (DOPC) lipid bilayers that are only electrostatically bound to the nanorod surface. The DOPC bilayer structure was confirmed by analyzing the position and orientation of the carbon double bond stretch relative to the symmetric choline vibration in the headgroup. Tryptophan was introduced into the DOPC bilayer, and through analysis of three of its vibrational modes the tryptophan position and orientation were determined. The orientation was in good agreement with NMR and fluorescence measurements, while the position was found to be somewhat deeper into the acyl chain region of the lipid bilayer. This method offers a new approach to analyzing lipid membrane molecular structure under ambient conditions and without molecular labels.

Multiple spectroscopic studies on the interaction of BSA with pristine CNTs and their toxicity against Donax faba

Enhanced biomedical applications of carbon nanotubes (CNTs) have necessitated the need for the fundamental understanding behind their interaction with biomolecules. Carbon nanotube bundles were dispersed with tween 20 to attain individual nanotubes that are active in UV–vis region. Plasmon resonance of dispersions was obtained at 252nm of 4.88eV. FT-Raman bands of dispersions of SWCNTs and MWCNTs at 1346 and 1573cm−1 corresponds to the existence of Disordered and Graphitic band of CNTs after dispersion. XRD revealed crystalline feature of graphitic structures of CNTs. SEM-EDAX showed tubular structures of CNTs along with the existence of lower percentage contents of metal impurities. Hyperchromic effect of BSA–CNT complex suggested the existence of ground state complex between them. Quenching nature of CNTs against the intrinsic fluorescence potential of BSA followed static mechanism. 3D fluorescence spectra of BSA confirmed the possibilities of their binding with CNTs via tryptophan and tyrosine residues, followed by the disturbances to their aromatic environment. Alterations were observed in the amide band position of FTIR spectra of BSA–CNT conjugates. Loss of alpha-helical structures and alteration in the tryptophan position were evidenced with respect to CD spectra. The toxicity profile of pristine and BSA–CNT conjugates were evaluated against Donax faba. On comparison with pristine CNTs, BSA–CNTs conjugates showed higher LC50 dose values. In addition, the histopathology of the tissues, treated with CNT–BSA conjugates has shown decreased effect on the cellular integrity rather than the pristine ones. Hence, the adsorption of biomolecules such as the proteins over CNTs surface could have possible effect on reducing their toxicity profile in nature.

Comprehensive spectroscopic studies on the interaction of biomolecules with surfactant detached multi-walled carbon nanotubes

This paper investigates the interaction of ten diverse biomolecules with surfactant detached Multi-Walled Carbon Nanotubes (MWCNTs) using multiple spectroscopic methods. Declining fluorescence intensity of biomolecules in combination with the hyperchromic effect in UV-Visible spectra confirmed the existence of the ground state complex formation. Quenching mechanism remains static and non-fluorescent. 3D spectral data of biomolecules suggested the possibilities of disturbances to the aromatic microenvironment of tryptophan and tyrosine residues arising out of CNTs interaction. Amide band Shifts corresponding to the secondary structure of biomolecules were observed in the of FTIR and FT-Raman spectra. In addition, there exists an increased Raman intensity of tryptophan residues of biomolecules upon interaction with CNTs. Hence, the binding of the aromatic structures of CNTs with the aromatic amino acid residues, in a particular, tryptophan was evidenced. Far UV Circular spectra have showed the loss of alpha-helical contents in biomolecules upon interaction with CNTs. Near UV CD spectra confirmed the alterations in the tryptophan positions of the peptide backbone. Hence, our results have demonstrated that the interaction of biomolecules with OH-MWCNTs would involve binding cum structural changes and alteration to their aromatic micro-environment.

Contrasting effects of W781V and W780V mutations in helix N of herpes simplex virus 1 and human cytomegalovirus DNA polymerases on antiviral drug susceptibility.

DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC50s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC50) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45- and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains. DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.

Site-selective C(sp3)-H functionalization of di-, tri-, and tetrapeptides at the N-terminus.

Although the syntheses of novel and diverse peptides rely mainly on traditional coupling using unnatural amino acids, postsynthetic modification of peptides could provide a complementary method for the preparation of nonproteinogenic peptides. Site selectivity of postsynthetic modification of peptides is usually achieved by targeting reactive moieties, such as the thiol group of cysteine or the C-2 position of tryptophan. Herein, we report the development of site-selective functionalizations of inert C(sp3)–H bonds of N-terminal amino acids in di-, tri-, and tetrapeptides without installing a directing group. The native amino acid moiety within the peptide is used as a ligand to accelerate the C–H activation reaction. In the long run, this newly uncovered reactivity could provide guidance for developing site-selective C(sp3)–H activation toward postsynthetic modification of a broader range of peptides.

Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB

LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal α-helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp(25)) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves.

Investigation of membrane penetration depth and interactions of the amino-terminal domain of huntingtin: refined analysis by tryptophan fluorescence measurement.

The membrane-association properties of the amino-terminal domain of huntingtin are accompanied by subcellular redistribution of the protein in cellular compartments. In this study we used tryptophan substitution of amino-acid residues at different positions of the huntingtin 1-17 domain (Htt17) to precisely determine, for the first time, the depth of penetration of the peptides within the lipid bilayer. Initially, secondary structure preferences and membrane association properties were quantitatively determined for several membrane lipid compositions; they were found to be closely related to those of the natural peptide, indicating that changes in the sequence had little effect on these characteristics of the domain. The tryptophan-substituted peptides became inserted into the membranes' interfacial region, with average tryptophan positions between 7.5 and 11Å from the bilayer center, in agreement with in-plane orientation of the peptide. Participation of the very-amino terminus of the peptide in the membrane-association process was demonstrated. The results not only revealed the occurrence of association intermediates when the huntingtin 1-17 anchoring sequence became inserted into the membrane but also suggest the formation of aggregates and/or oligomers during membrane association. When inserted, the F11W site was of crucial importance in lipid anchoring and stabilization of the whole peptide, whereas the terminal residues are located close to the membrane surface. The carboxy-terminal tryptophan (F17W), which also constitutes the site of the polyglutamine extension in the natural domain, was found closest to the aqueous environment, accompanied with the highest aqueous quenching constants. These results were used to propose a refined model of lipid interactions of the huntingtin 1-17 domain.

Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-014-0958-9) contains supplementary material, which is available to authorized users.

ChemInform Abstract: Direct Olefination at the C‐4 Position of Tryptophan via C—H Activation: Application to Biomimetic Synthesis of Clavicipitic Acid.

AbstractThe reaction proceeds under mild conditions with various substituted olefins to give the corresponding 4‐substituted tryptophans.

Direct Olefination at the C-4 Position of Tryptophan via C–H Activation: Application to Biomimetic Synthesis of Clavicipitic Acid

The first Pd-catalyzed method for direct olefination at the C4 position of tryptophan derivatives has been developed via C-H activation to prepare 4-substituted tryptophans, which could be used for the synthesis of many hemiterpenoid indole alkaloids. This reaction proceeds under mild reaction conditions and with exceptional tolerance to a variety of functional groups. Furthermore, the efficiency of this method is demonstrated by the rapid and biomimetic synthesis of clavicipitic acid.

Large Scale Dynamics of the Michaelis Complex in Bacillus stearothermophilus Lactate Dehydrogenase Revealed by a Single-Tryptophan Mutant Study

Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophilic lactate dehydrogenase, bsLDH·NADH·oxamate, were studied with site specific resolution by laser-induced temperature jump relaxation spectroscopy with a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the millisecond to microsecond time scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called "loop motion" is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on the catalytic mechanism of bsLDH is discussed.

Arginine171 of Chlamydomonas reinhardtii [Fe–Fe] hydrogenase HydA1 plays a crucial role in electron transfer to its catalytic center

[Fe–Fe] hydrogenases, with hydrogen evolution activities outperforming [Ni–Fe] hydrogenases by 3–4 orders of magnitude, are still the most promising enzyme class for hydrogen production purposes. For Chlamydomonas reinhardtii [Fe–Fe] hydrogenase HydA1 the question of catalytic activity and electron transport is of main importance. Here we report the characterization of two mutant forms of C. reinhardtii HydA1. An aspartic acid in place of arginine171 leads to a six-fold increase of the catalytic activity in comparison to the wild type protein during methyl viologen-dependent hydrogen production. Tryptophan in position 171 does not result in any change in methyl viologen-induced activity. At the same time these mutations lead to a strong decrease in ferredoxin-dependent hydrogen production while the catalytic center of mutant forms stays intact. The localization of this amino acid (arginine171) in the environment of CrHydA1 H-cluster indicates that the limitation of the catalytic activity of this hydrogenase is due to the electron transfer step to the catalytic center where the reduction of protons takes place.

HIV-1 genome is often defective in PBMCs and rectal tissues after long-term HAART as a result of APOBEC3 editing and correlates with the size of reservoirs

Precise characterization of viruses present in reservoirs in long-term pretreated patients will be a major issue to consider in the context of viral eradication. We assessed the frequency of defective viruses present in cellular reservoirs. Peripheral blood mononuclear cells (PBMCs) and rectal biopsy samples were compared between five patients on successful long-term highly active antiretroviral therapy (HAART) (>7 years without blips) and five untreated patients. Molecular cloning and sequencing of the reverse transcriptase region were used to detect the presence of and quantify in-frame stop codons in HIV quasi-species. The relationship between the size of the reservoir and the frequency of defective genomes was assessed. Defective genomes were systematically detected in all patients on long-term HAART in both compartments (PBMCs and rectal tissues), with a higher level of defective genomes per sample compared with PBMCs of untreated patients. A high level of defective genomes was correlated with a small size of HIV proviral DNA. Regarding the nucleotide context, guanine (G) to adenine (A) substitution at tryptophan positions was responsible for the appearance of 89% of all in-frame stop codons in the context of G-to-A hypermutation, likely reflecting APOBEC3 footprints on the viral genome. We propose a scenario whereby defective genomes accumulate during HAART treatment, eventually reaching a viral extinction threshold. In the context of viral eradication, measurement of the relative amounts of defective and non-defective viruses (by molecular cloning and ultradeep sequencing) should be used as a new criterion for eradicating HIV.

Quantitative evaluation of tryptophan oxidation in actin and troponin I from skeletal muscles using a rat model of acute oxidative stress

Increased levels of "ROS" cause oxidative stress and are believed to play a key role in the development of age-related diseases and mammalian aging, e.g. through the oxidation of residues, at or close to, the protein surface. In this study, we have investigated the effects of ROS on tryptophan residues in alpha skeletal actin and troponin I (fast skeletal muscle isoform) using an established rat model of acute oxidative stress induced by X-ray irradiation. In the control samples (no oxidative stress), the single Trp residue of troponin I (position 161) and the four tryptophan residues present in actin (positions 79, 86, 340, and 356) were only oxidized at very low levels. Post-irradiation, the level of oxidized versions increased for most positions within 3 h. Tryptophan residues located inside the proteins, however, required longer time periods. Based on the increment masses of the tryptophan positions calculated from the b- and y-ion series of the tandem mass spectra, the following oxidation products of tryptophan were detected: kynurenine; oxolactone; hydroxytryptophan or oxindolylalanine (isobaric); hydroxykynurenine; dioxindolylalanine, N-formylkynurenine or dihydroxytryptophan (all three isobaric); and hydroxyl-N-formylkynurenine, with mass gains relative to tryptophan of 4, 14, 16, 20, 32, and 48 u, respectively. Despite a partial recovery after 24 h, the degree of oxidation of all oxidized versions was still higher than in the control samples.

Expression of Bv8 in Pichia pastoris to identify structural features for receptor binding

Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.

A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors

The conserved tryptophan in position 13 of TM-VI (Trp-VI:13 or Trp-6.48) of the CWXP motif located at the bottom of the main ligand-binding pocket in TM-VI is believed to function as a rotameric microswitch in the activation process of seven-transmembrane (7TM) receptors. Molecular dynamics simulations in rhodopsin demonstrated that rotation around the chi1 torsion angle of Trp-VI:13 brings its side chain close to the equally highly conserved Phe-V:13 (Phe-5.47) in TM-V. In the ghrelin receptor, engineering of high affinity metal-ion sites between these positions confirmed their close spatial proximity. Mutational analysis was performed in the ghrelin receptor with multiple substitutions and with Ala substitutions in GPR119, GPR39, and the beta(2)-adrenergic receptor as well as the NK1 receptor. In all of these cases, it was found that mutation of the Trp-VI:13 rotameric switch itself eliminated the constitutive signaling and strongly impaired agonist-induced signaling without affecting agonist affinity and potency. Ala substitution of Phe-V:13, the presumed interaction partner for Trp-VI:13, also in all cases impaired both the constitutive and the agonist-induced receptor signaling, but not to the same degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency. In a proposed active receptor conformation generated by molecular simulations, where the extracellular segment of TM-VI is tilted inwards in the main ligand-binding pocket, Trp-VI:13 could rotate into a position where it obtained an ideal aromatic-aromatic interaction with Phe-V:13. It is concluded that Phe-V:13 can serve as an aromatic lock for the proposed active conformation of the Trp-VI:13 rotameric switch, being involved in the global movement of TM-V and TM-VI in 7TM receptor activation.