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Context: Riboflavin (RF), also known as B2 vitamin, is the precursor to flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), two co-enzymes involved in many electron transport processes. Interactions of the isoalloxazine ring, common to all three compounds, are of great interest due to their biological function in flavoproteins and relevance in the transport by the carrier protein leading to development of drug delivery strategies and non-invasive diagnostics techniques. Based on protein crystallographic data, a computational investigation of the interactions in the complexes between lumiflavin, a model compound, and aromatic amino acids, tyrosine and tryptophan, was pursued with the goal of characterizing noncovalent interactions. Density functional theory (DFT) served as the computation framework for all calculations, utilizing long-range corrected hybrid functionals LC-ωPBE and ωB97XD in conjunction with the 6-311+ +g** basis set. The solvation effects were incorporated through the implementation of the polarizable continuum model (PCM) simulating an aqueous solvent environment. The geometries of the five most stable complexes show exclusively p-p interactions among the aromatic moieties in a displaced parallel plane stacking arrangement with interplanar heights and displacements in the range of 3.22-3.62 Å and 0.50-0.63 Å, respectively, at ωB97XD level. The calculated total energies and binding energies indicate two stabilizing p-p interactions: lumiflavin-tyrosine and lumiflavin-tryptophan, with the later stronger for the more stable complexes by 2 kcal mol -1 . The complexes are less entropically favored than the independent molecules as verified by the positive association free Gibbs energies with LC-ωPBE and nearly zero with ωB97XD. Orbital analysis indicates a smaller HOMO-LUMO gap for complexes compared to the individual compounds suggesting a charge transfer component to the interaction. Moreover, the HOMO is localized on tryptophan and HOMO-1 on tyrosine, consistent with the strength of the respective interactions with lumiflavin., Methods: The initial geometry was based on the atom coordinates of the bonding tryptophan-riboflavin-tyrosine region in the protein crystallographic data with the ribityl tail being discarded, leading to a model complex: tryptophan-lumiflavin-tyrosine. The initial conformational search using the Amber force field within the Gabedit led to 30 unique conformations. The subsequent calculations, energy optimization and orbital analysis, were performed in Guassian16 at density functional theory (DFT) level, utilizing long-range corrected hybrid functionals LC-ωPBE and ωB97XD in conjunction with the 6-311+ +g** basis set. The solvent, water, was accounted for using the polarized continuum model (PCM)., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The recent application of 19 F NMR in the study of biomolecular structure and dynamics has made it a potentially attractive probe to complement traditional 15 N/ 13 C labelled probes for backbone and sidechain dynamics, albeit with some complications. The utility of 15 N relaxation rates of rigid backbone amide groups to determine the rotational diffusion tensor of proteins is well established. Here we show that the measured 19 F relaxation rates of two buried and possibly immobile 19 F labelled tryptophan sidechains for the multidomain protein RfaH, in its closed conformation, are in reasonable agreement with the calculated values, only when anisotropic rotational diffusion of the protein is considered. While the sparsity of 19 F relaxation data from a limited number of probes precludes the experimental determination of the rotational diffusion tensor here, these results demonstrate the influence of rotational diffusion anisotropy of proteins on 19 F NMR relaxation of rigid tryptophan sidechains, while adding to the expanding literature of 19 F NMR relaxation data sets in biomolecules., Competing Interests: Declarations. Competing interests: The authors declare no competing interests, (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19 F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19 F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A 5-day test duration makes BOD 5 measurement unsatisfactory and hinders the development of a quick technique. Protein-like fluorescence peaks show a strong correlation between the BOD characteristics and the fluorescence intensities. For identifying and measuring BOD in surface water, a simultaneous absorbance-transmittance and fluorescence excitation-emission matrices (A-TEEM) method combined with PARAFAC (parallel factor) and PLS (partial least squares) analyses was developed using a tyrosine and tryptophan (tyr-trpt) mix as a surrogate analyte for BOD. The use of a surrogate analyte was decided upon due to lack of fluorescent BOD standards. Tyr-trpt mix standard solutions were added to surface water samples to prepare calibration and validation samples. PARAFAC analysis of excitation-emission matrices detected the tyr-trpt mix in surface water. PLS modelling demonstrated significant linearity (R 2  = 0.991) between the predicted and measured tyr-trypt mix concentrations, and accuracy and robustness were all acceptable per the ICH Q2 (R2) and ASTM multivariate calibration/validation procedures guidelines. Based on a suitable and workable surrogate analyte method, these results imply that BOD can be detected and quantified using the A-TEEM-PARAFAC-PLS method. Very positive comparability between tyr-trypt mix concentrations was found, suggesting that tyr-trypt mix might eventually take the place of a BOD-based sampling protocol. Overall, this approach offers a novel tool that can be quickly applied in water treatment plant settings and is a step in supporting the trend toward rapid BOD determination in waters. Further studies should demonstrate the wide application of the method using real wastewater samples from various water treatment facilities., (© 2024. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15 N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered., (© 2022. The Author(s).)

The structural organization and dynamic nature of the biomembrane components are important determinants for numerous cellular functions. Particularly, membrane proteins are critically important for various physiological functions and are important drug targets. The mechanistic insights on the complex functionality of membrane lipids and proteins can be elucidated by understanding the interplay between structure and dynamics. In this regard, membrane penetration depth represents an important parameter to obtain the precise depth of membrane-embedded molecules that often define the conformation and topology of membrane probes and proteins. In this review, we discuss about the widely used fluorescence quenching-based methods (parallax method, distribution analysis, and dual-quencher analysis) to accurately determine the membrane penetration depths of fluorescent probes that are either membrane-embedded or attached to lipids and proteins. Further, we also discuss a relatively novel fluorescence quenching method that utilizes tryptophan residue as the quencher, namely the tryptophan-induced quenching, which is sensitive to monitor small-scale conformational changes (short distances of < 15 Å) and useful in mapping distances in proteins. We have provided numerous examples for the benefit of readers to appreciate the importance and applicability of these simple yet powerful methods to study membrane proteins., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Selectivity of dopamine (DA), uric acid (UA), and ascorbic acid (AA) is an open challenge of electrochemical sensors in the field of biosensing. In this study, two selective mechanisms for detecting DA, UA, and AA biomolecules on the pristine boron nitride nanosheets (BNNS) and functionalized BNNS with tryptophan (Trp), i.e., Trp@BNNS have been illustrated through density functional density (DFT) calculation and charge population analysis. Our findings reveal that the adsorbed biomolecules on Trp@BNNS indicate the less sensitivity factor of biomolecule separation than the functionalized biomolecules with Trp (Trp@biomolecule) adsorbed on pristine BNNS. From the calculations, strong adsorption of Trp@biomolecule on the pristine substrate corresponds to enhancing of electron charge transfer and electrical dipole moment. Our analysis is in good agreement with the previous theoretical and experimental results and suggests new pathway for electrode modification for electrochemical biosensing., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Preeclampsia (PE) is a hypertensive pregnancy, which is a leading cause of maternal and fetal morbidity and mortality during pregnancy. L-Tryptophan (Trp) is an essential amino acid, which can be metabolized into various biologically active metabolites. However, the levels of many circulating Trp-metabolites in human normotensive pregnancies (NT) and PE are undetermined. This study quantified the levels of Trp-metabolites in maternal and umbilical vein sera from women with NT and PE. Paired maternal and umbilical blood samples were collected from singleton pregnant patients. Twenty-five Trp-metabolites were measured in serum samples using liquid chromatography with tandem mass spectrometry. The effects of L-kynurenine (Kyn) and indole-3-lactic acid (ILA), on function of human umbilical vein endothelial cells (HUVECs), were also determined. Twenty Trp-metabolites were detected. The levels of 9 Trp-metabolites including Kyn and ILA were higher (P < 0.05) in umbilical vein than maternal serum, whereas 2 (5-hydroxy-L-tryptophan and serotonin) were lower (P < 0.05) in umbilical vein compared to maternal serum. PE significantly (P < 0.05) elevated ILA levels in maternal and umbilical vein sera. Kyn dose-dependently decreased (P < 0.05) cell viability. Kyn and ILA dose- and time-dependently (P < 0.05) increased monolayer integrity in HUVECs. These data suggest that these Trp-metabolites are important in regulating endothelial function during pregnancy, and the elevated ILA in PE may antagonize increased endothelial permeability occurring in PE., (© 2021. Society for Reproductive Investigation.)

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100 ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950 ps and ~ 50 ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.

Tryptophan is an amino acid required by all life forms for protein synthesis and other important metabolic functions. It is metabolized in the body using the kynurenine pathway which involves the enzyme indoleamine 2,3 dioxygenase (IDO) and its transport is regulated through the L-type amino acid transporters (LAT 1). IDO and LAT 1 are found to be overexpressed in many cancers i.e., ovarian, lung colorectal etc. In this study we have used this specific interaction as the basis for designing diagnostic agent based on iron oxide nanoparticles which can specifically target the IDO/LAT 1 over expressing tumors. We have conjugated tryptophan to the surface of super-paramagnetic nanoparticles chemically using 3-aminopropyltrimethoxysilane as a linker. The synthesized tryptophan conjugated magnetic nano-conjugate has been characterized using FTIR, UV-Vis, TEM for its shape size, charge and NMR and Mass for conjugation. The magnetization studies show decrease in the magnetic behavior after conjugation however the desired super-paramagnetic property is still retained as shown by the signature sigmoidal B-H curve. The nano-conjugate shows minimal cytotoxicity over 24 h as shown by the SRB assay in two cell lines A-549, MCF-7. Using 99mTc labeling the biodistribution and the blood kinetics of the magnetic nano-conjugate was evaluated. The study highlights the suitability of the designed magnetic Nano bioconjugate as a potential bimodal diagnostic agent.

C-Mannosyl tryptophan (C-Man-Trp) is a unique glycosylated amino acid present in various eukaryotes. The C-Man-Trp structure can be found as a monomeric form or a part of post-translational modifications within polypeptide chains in living organisms. However, the mechanism of how monomeric C-Man-Trp is produced has not been fully investigated. In this study, we assessed levels of cellular C-Man-Trp by ultra performance liquid chromatography with a mass spectrometry assay system, and investigated whether the cellular C-Man-Trp is affected by autophagy induction. The intracellular C-Man-Trp level was significantly increased under serum and/or amino acid starvation in A549, HaCaT, HepG2, NIH3T3, and NRK49F cells. The increase in C-Man-Trp was also observed in NIH3T3 cells treated with rapamycin, an autophagy inducer. The up-regulation of C-Man-Trp caused by starvation was reversed by the inhibition of lysosomal enzymes. We further showed that C-Man-Trp is produced by incubating a synthetic C-mannosylated peptide (C-Man-Trp-Ser-Pro-Trp) or thrombospondin (TSP) in a lysosomal fraction that was prepared from a mouse liver, which provides supporting evidence that C-Man-Trp is a degradation product of the C-mannosylated peptide or protein following lysosome-related proteolysis. Taken together, we propose that the autophagic pathway is a novel pathway that at least partly contributes to intracellular C-Man-Trp production under certain conditions, such as nutrient starvation.

Due to its metabolism via the serotonin and kynurenine pathways, tryptophan plays a key role in multiple disease processes including cancer. Imaging tryptophan uptake and metabolism in vivo can be achieved with tryptophan derivative positron emission tomography (PET) radiotracers. While human studies with such tracers have been confined to C-11-labeled compounds, preclinical development of F-18-labeled tryptophan-based radiotracers has surged in recent years. We performed a systematic review of studies reporting on such F-18-labeled tryptophan tracers to summarize and compare their biological characteristics and their potential for tumor imaging, with a particular focus on key enzymes of the kynurenine pathway (indoleamine 2,3-dioxygenase [IDO] and tryptophan 2,3-dioxygenase [TDO]), which play an important role in tumoral immune resistance. From a PubMed search, English language articles including data on the preparation and radiochemical and/or biological characteristics of F-18-labeled tryptophan derivative radiotracers were reviewed. A total of 19 original papers included data on 15 unique radiotracers, the majority of which were synthesized with an adequate radiochemical yield. Automated synthesis was reported for 1-(2-[ 18 F]fluoroethyl)-L-tryptophan, the most extensively evaluated tracer thus far. Biodistribution studies showed high uptake in the pancreas, while the L-type amino acid transporter was the dominant transport mechanism for most of the reviewed tracers. Tracers tested for tumor uptake showed accumulation in tumor cell lines in vitro and in xenografts in vivo, often with favorable tumor-to-background uptake ratios in comparison with clinically used F-18-labeled radiotracers. Five tracers showed promise for imaging IDO activity, including 1-(2-[ 18 F]fluoroethyl)-L-tryptophan and a F-18-labeled analog of alpha-[ 11 C]methyl-L-tryptophan tested clinically in previous studies. Two radiotracers were metabolized by TDO but showed defluorination in vivo. In summary, most F-18-labeled tryptophan derivative PET tracers share common transport mechanisms and biodistribution characteristics. Several reported tracers could be candidates for further testing and validation toward PET imaging applications in a variety of human diseases.

A fluorescence quenching study of the fluorescent probes L-tryptophan and indole by anions (NO 3 - , Cl - , SO 4 2- ) was carried out in an aqueous system. The ions NO 3 - , Cl - and SO 4 2- showed very good quenching of both the probes. Quenching of L-tryptophan by all the anions studied was higher as compare to the quenching of indole. The data was fitted with the Stern-Volmer equation. Stern-Volmer constants were observed in the order NO 3 - > Cl - > SO 4 2- . Stern-Volmer constants reflect the sensitivity of the method for the studied anions. Limit of detection (LOD) was calculated as three times the standard deviation of the blank for n = 10 (3 × SD) while the limit of quantification (LOQ) was calculated as ten times the standard deviation of the blank for n = 10 (10 × SD). In the case of L-tryptophan LOD and LOQ varied from 4.08 × 10 -5 - 4.56 × 10 -4 mol L -1 , while in the case of indole the values ranged from 3.87 × 10 -5 - 6.59 × 10 -4 respectively. Fluorescence quenching of L-tryptophan and indole by the studied anions showed good reproducibility and the method could be very effective for the determination of anions.

A novel zinc(II)-salphen-azo-benzo-15-crown-5 triad receptor was studied theoretically as ditopic recognition of hydrophobic amino acid. And density functional theory was used to investigate the Zn(II) salphen-crown ether complex (L 1 ), complex L 2 (L 1 with the alkaline metal cation Na + ), and the corresponding 1:1 sandwich complex (complex L with tryptophan). In this work, geometrical optimization was carried out using ωB97XD functional and def2-SVP basis set for all atoms. The absorption spectra and excited-states were calculated using time-dependent density functional theory and ωB97XD/def2-TZVP level. The absorption spectra data show some significantly shifts in the absorption band due to the present of Na + or tryptophan. In addition, interfragment interactions between receptor L and tryptophan were analyzed in detail by Independent Gradient Model and topological properties of Bader's atoms in molecules theory, which is found to contribute to forming the metal-ligand bonds, intermolecular H-bond, and van der Waals interaction in 1:1 sandwich complex. The above results demonstrate that the L 2 complex is a ditopic receptor to be utilized to recognize amphiphilic molecule - tryptophan.

Purpose: Fluorine-18 labeled tryptophan analog L-1-[ 18 F]fluoroethyl-tryptophan (L-1-[ 18 F]FETrp) was designed for positron emission tomography (PET) imaging of cancer by dual targeting of the overexpressed amino acid transporters and altered indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway of tryptophan metabolism. In our previous study, we described the radiosynthesis and preliminary evaluation of L-1-[ 18 F]FETrp for PET imaging of breast cancer. The aim of this study was to investigate the in vivo imaging mechanism and further evaluate this radiotracer in more wide range types of cancers including prostate cancer, lung cancer, and glioma., Procedures: The mice bearing subcutaneous PC-3 prostate cancer, subcutaneous H2009 and H460 lung cancers, subcutaneous MDA-MB-231, orthotopic A549 lung cancer, and intracranial 73C glioma were employed to evaluate L-1-[ 18 F]FETrp for PET imaging of cancer. The in vivo catabolism of L-1-[ 18 F]FETrp in the tumor was studied by analysis of PC-3 extracts with radio-HPLC., Results: Small animal PET/CT imaging of L-1-[ 18 F]FETrp visualized all tumors in these different mouse models with high accumulations of radioactivity in PC-3 (7.5 ± 0.6 % ID/g), H2009 (5.3 ± 0.8 % ID/g), H460 (9.0 ± 1.4 % ID/g), A549 (4.5 ± 0.5 % ID/g), and 73C (4.1 ± 0.7 % ID/g) tumors. The radio-HPLC analysis of PC-3 tumor extracts revealed that about 30 % of L-1-[ 18 F]FETrp was converted into a highly polar radioactive metabolite. The uptake in H460 cancer was about 1.7-fold higher than that in H2009 cancer, which indicated L-1-[ 18 F]FETrp could differentiate these subtypes of lung cancers (H2009 and H460) by imaging quantification. Furthermore, small animal PET/CT imaging in intracranial glioma revealed L-1-[ 18 F]FETrp could pass blood-brain barrier (BBB) and accumulate in glioma with a favorable imaging contrast (tumor-to-brain 2.9)., Conclusions: L-1-[ 18 F]FETrp highly accumulated in a wide range of malignancies including lung cancer, prostate cancer, and glioma. These results suggested that L-1-[ 18 F]FETrp is a promising radiotracer for PET imaging of cancer.

We present 19 F longitudinal and transverse relaxation studies for four differently fluorosubstituted L-tryptophans, which carry single F atoms in the indole ring, both in the context of the free amino acid and when located in the cyclophilin A protein. For the free 4F-, 5F-, 6F-, 7F-L-Trp, satisfactory agreement between experimentally measured and calculated relaxation rates was obtained, suggesting that the parameters used for calculating the rates for the indole frame are sufficiently accurate. We also measured and calculated relaxation rates for four differently 19 F-tryptophan labeled cyclophilin A proteins, transferring the parameters from the free amino acid to the protein-bound moiety. Our results suggest that 19 F relaxation data of the large and rigid indole ring in Trp are only moderately affected by protein motions and provide critical reference points for evaluating fluorine NMR relaxation in the future, especially in fluorotryptophan labeled proteins.

The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the heating of pre-frozen to - 180 °C in the dark or in the light-activated preparations has been studied. Fluorescence lifetime in samples frozen in the light was longer than in samples frozen in the dark. For samples in 65% glycerol at λ reg  = 335 nm and at 20 °C, the lifetime of components were as follows: τ 1  ≈ 1.2 ns, τ 2  ≈ 4.9 ns, and τ 3  ≈ 20 ns. The contribution of the first component was negligible. To analyze the contribution of components 2 and 3 derived from frozen-thawed samples, two temperature ranges from - 180 to - 90 °C and above - 90 °C are considered. In doing so, the contributions of these components appear antiphase course to each other. The dependence on temperature of these contributions is explained by the influence of the microconformational protein dynamics on the tryptophan fluorescence lifetime. In the present work, a comparative analysis of temperature-dependent conformational dynamics and electron transfer in cyanobacterial PSI (Schlodder et al., in Biochemistry 37:9466-9476, 1998) and Rhodobacter sphaeroides reaction center complexes (Knox et al., in J Photochem Photobiol B 180:140-148, 2018) was also carried out.

Organic matter (OM), a complex entity with diverse functional groups and molecular sizes, has important effects on aquatic systems. We studied the optical compositions and sources of dissolved organic matter (DOM) and particulate organic matter (POM) in Lake Taihu, a large, shallow and eutrophic lake in China. Significant differences in optical compositions and sources occurred between the POM and DOM. The temporal-spatial distribution of the fluorescence indices suggested that the POM in Lake Taihu was mainly from autochthonous sources, but more exogenous characteristics were shown in POM in the river mouths compared with other regions. The chromophoric DOM in Lake Taihu mainly displayed autochthonous characteristics. The POM-DOM PARAFAC model was used to examine OM optical composition and five components were identified, which contained three protein-like components (C1, C2, and C5), a microbial humic-like component (C3), and a terrestrial humic-like component (C4). The POM was dominated by C5 in summer and autumn and C3 in winter and spring, and the DOM was dominated by protein-like components (C1, C2, and C5) through the entire year. The algae-dominated region had a relative higher contribution of tryptophan-like components of POM compared with the macrophyte-dominated region. A conceptual model based on the theory of "four phases of cyanobacteria bloom development" was proposed to fully describe the relationship between POM-DOM exchanges and cyanobacteria bloom development.

In contrast to the parent pentadecapeptide gramicidin A (gA), some of its cationic analogs have been shown previously to form large-diameter pores in lipid membranes. These pores are permeable to fluorescent dyes, which allows one to monitor pore formation by using the fluorescence de-quenching assay. According to the previously proposed model, the gA analog with lysine substituted for alanine at position 3, [Lys3]gA, forms pores by a homopentameric assembly of gramicidin double-stranded β-helical dimers. Here, we studied the newly synthesized analogs of [Lys3]gA with single, double and triple substitutions of isoleucines for tryptophans at positions 9, 11, 13, and 15. Replacement of any of the tryptophans of [Lys3]gA with isoleucine resulted in suppression of the pore-forming activity of the peptide, the effect being significantly dependent on the position of tryptophans. In particular, the peptide with a single substitution of tryptophan 13 showed much lower activity than the analogs with single substitutions at positions 9, 11, or 15. Of the peptides with double substitutions, the strongest suppression of the leakage was observed with tryptophans 13 and 15. In the case of triple substitutions, only the peptide retaining tryptophan 11 exhibited noticeable activity. It is concluded that tryptophans 11 and 13 contribute most to pore stabilization in the membrane, whereas tryptophan 9 is not so important for pore formation. Cation-π interactions between the lysine and tryptophan residues of the peptide are suggested to be crucial for the formation of the [Lys3]gA pore.

Low-pressure (LP) UV treatment after chlorine disinfection was associated with enhanced formation of trichloronitromethane (TCNM), a halonitromethane disinfection by-product (DBP), due to the chlorination of tryptophan. Evidence was found that the concentration of TCNM from tryptophan increased quickly to the maximum for the first instance. Moreover, the increase of TCNM under UV exceeded 10 times than under dark. Then, it was found to have an obvious decrease in the formation of TCNM, even finally hardly disappear. In order to elucidate reasons for this phenomenon, the effects of light intensity, initial tryptophan concentration, free chlorine concentration, pH, and tert-butanol (TBA) on the formation of TCNM were investigated under UV/chlorine treatment. Finally, the effects of tryptophan on the formation of TCNM and the direct photodegradation of TCNM under LP UV irradiation were studied for analyzing the possible pathways of TCNM formation from amino acid. Since amino acids are very common in water sources, further research into chemical oxidation of these species by LP UV and chlorine is recommended. It can help us to find the precursors of TCNM formation and reduce the risk of TCNM formation for drinking water and wastewater utilities.

The insertion of suitable metals or high Z elements in tumour-avid tetrapyrrole compounds is a promising approach to obtain potential agents for multimodal cancer therapeutics and tumour imaging. Using chlorin p 6 , a chlorophyll derivative, we synthesized a novel iodinated chlorin p 6 -copper complex (ICp 6 -Cu) that can be applied to the photodynamic therapy and photon activation therapy of cancer. In the present study, we investigated the interaction of ICp 6 -Cu with human serum albumin (HSA) using UV-Vis absorption and fluorescence spectroscopy. The addition of HSA to ICp 6 -Cu at physiological pH led to a ∼7 nm red shift in its Soret and Q band absorption. The binding constant (K b ) and the number of binding sites (n) of ICp 6 -Cu obtained from the quenching of the intrinsic fluorescence of HSA were 2.9 × 10 6 M -1 and 1.2 respectively. The distance between the Trp-214 residue and ICp 6 -Cu computed from Förster non-radiative energy transfer (FRET) theory was 3.1 nm. The emission of the ICp 6 -Cu fluorescence when excited at the protein's Trp absorption (295 nm) further substantiated the FRET between the Trp residue and ICp 6 -Cu. Synchronous spectroscopy revealed that the quenching of protein Trp fluorescence was higher than that of Tyr with no significant shift in peak position. Results suggested that HSA acts as a carrier protein for ICp 6 -Cu with a high probability that the binding of ICp 6 -Cu occurred at subdomain IIA and the binding had no effect on the conformation of HSA.

The application of metabolic precursors for selective stable isotope labeling of aromatic residues in cell-based protein overexpression has already resulted in numerous NMR probes to study the structural and dynamic characteristics of proteins. With anthranilic acid, we present the structurally simplest precursor for exclusive tryptophan side chain labeling. A synthetic route to 13 C, 2 H isotopologues allows the installation of isolated 13 C- 1 H spin systems in the indole ring of tryptophan, representing a versatile tool to investigate side chain motion using relaxation-based experiments without the loss of magnetization due to strong 1 J CC and weaker 2 J CH scalar couplings, as well as dipolar interactions with remote hydrogens. In this article, we want to introduce this novel precursor in the context of hitherto existing techniques of in vivo aromatic residue labeling.

Experimental studies on protein dynamics at atomic resolution by NMR-spectroscopy in solution require isolated 1 H-X spin pairs. This is the default scenario in standard 1 H- 15 N backbone experiments. Side chain dynamic experiments, which allow to study specific local processes like proton-transfer, or tautomerization, require isolated 1 H- 13 C sites which must be produced by site-selective 13 C labeling. In the most general way this is achieved by using site-selectively 13 C-enriched glucose as the carbon source in bacterial expression systems. Here we systematically investigate the use of site-selectively 13 C-enriched ribose as a suitable precursor for 13 C labeled histidines and tryptophans. The 13 C incorporation in nearly all sites of all 20 amino acids was quantified and compared to glucose based labeling. In general the ribose approach results in more selective labeling. 1- 13 C ribose exclusively labels His δ2 and Trp δ1 in aromatic side chains and helps to resolve possible overlap problems. The incorporation yield is however only 37% in total and 72% compared to yields of 2- 13 C glucose. A combined approach of 1- 13 C ribose and 2- 13 C glucose maximizes 13 C incorporation to 75% in total and 150% compared to 2- 13 C glucose only. Further histidine positions β, α and CO become significantly labeled at around 50% in total by 3-, 4- or 5- 13 C ribose. Interestingly backbone CO of Gly, Ala, Cys, Ser, Val, Phe and Tyr are labeled at 40-50% in total with 3- 13 C ribose, compared to 5% and below for 1- 13 C and 2- 13 C glucose. Using ribose instead of glucose as a source for site-selective 13 C labeling enables a very selective labeling of certain positions and thereby expanding the toolbox for customized isotope labeling of amino-acids.

The fluorescence quenching, by a series of amino acids, of pseudopeptidic compounds acting as probes for cellular acidity has been investigated. It has been found that amino acids containing electron-rich aromatic side chains like Trp or Tyr, as well as Met quench the emission of the probes mainly via a collisional mechanism, with Stern-Volmer constants in the 7-43 M -1 range, while other amino acids such as His, Val or Phe did not cause deactivation of the fluorescence. Only a minor contribution of a static quenching due to the formation of ground-state complexes has been found for Trp and Tyr, with association constants in the 9-24 M -1 range. For these ground-state complexes, a comparison between the macrocyclic probes and an open chain analogue reveals the existence of a moderate macrocyclic effect due to the preorganization of the probes in the more rigid structure.

Construction of predominantly one-handed helical polyacetylenes with a desired helix sense utilizing noncovalent chiral interactions with nonracemic chiral guest compounds based on a supramolecular approach is described. As with the conventional dynamic helical polymers possessing optically active pendant groups covalently bonded to the polymer chains, this noncovalent helicity induction system can show significant chiral amplification phenomena, in which the chiral information of the nonracemic guests can transfer with high cooperativity through noncovalent bonding interactions to induce an almost single-handed helical conformation in the polymer backbone. An intriguing "memory effect" of the induced macromolecular helicity is observed for some polyacetylenes, which means that the helical conformations induced in dynamic helical polyacetylene can be transformed into metastable static ones by tuning their helix-inversion barriers. Potential applications of helical polyacetylenes with controlled helix sense constructed by the "noncovalent helicity induction and/or memory effect" as chiral materials are also described.

Purpose: Tryptophan metabolism via indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway plays a role in immunomodulation and has been emerging as a plausible target for cancer immunotherapy. Imaging IDO-mediated kynurenine pathway of tryptophan metabolism with positron emission tomography (PET) could provide valuable information for noninvasive assessment of cancer immunotherapy response. In this work, radiotracer 1-(2-[ 18 F]fluoroethyl)-L-tryptophan (1-L-[ 18 F]FETrp) and its enantioisomer 1-D-[ 18 F]FETrp were synthesized and evaluated for PET imaging of IDO-mediated kynurenine pathway of tryptophan metabolism., Procedures: Enantiopure 1-L-[ 18 F]FETrp and 1-D-[ 18 F]FETrp were prepared by a nucleophilic reaction of N-boc-1-(2-tosylethyl) tryptophan tert-butyl ester with [ 18 F]Fluoride, followed by acid hydrolysis in a GE Tracerlab FX-N module. In vitro cell uptake assays were performed with a breast cancer cell line MDA-MB-231. Small animal PET/computed tomography (CT) imaging was carried out in a mouse model bearing MDA-MB-231 xenografts., Results: Automatic radiosynthesis of 1-L-[ 18 F]FETrp and 1-D-[ 18 F]FETrp was achieved by a one-pot two-step approach in 19.0 ± 7.0 and 9.0 ± 3.0 % (n = 3) decay-corrected yield with radiochemical purity over 99 %, respectively. In vitro cell uptake study indicated the uptake of 1-D-[ 18 F]FETrp in MDA-MB-231 cells was 0.73 ± 0.07 %/mg of protein at 60 min, while, the corresponding uptake of 1-L-[ 18 F]FETrp was 6.60 ± 0.77 %/mg. Further mechanistic assays revealed that amino acid transport systems L-tpye amino acid transporter (LAT) and alanine-, serine-, and cysteine-preferring (ASC), and enzyme IDO expression were involved in cell uptake of 1-L-[ 18 F]FETrp. Small animal PET/CT imaging study showed the tumor uptake of 1-L-[ 18 F]FETrp was 4.6 ± 0.4 % ID/g, while, the tumor uptake of 1-D-[ 18 F]FETrp was low to 1.0 ± 0.2 % ID/g, which were confirmed by ex vivo biodistribution study., Conclusions: We have developed a practical method for the automatic radiosynthesis of 1-L-[ 18 F]FETrp and 1-D-[ 18 F]FETrp. Our biological evaluation results suggest that 1-L-[ 18 F]FETrp is a promising radiotracer for PET imaging of IDO-mediated kynurenine pathway of tryptophan metabolism in cancer.

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein-ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery.

Drug-resistant microorganisms ('superbugs') present a serious challenge to the success of antimicrobial treatments. Subsequently, there is a crucial need for novel bio-control agents. Many antimicrobial peptides (AMPs) show a broad-spectrum activity against bacteria, fungi or viruses and are strong candidates to complement or substitute current antimicrobial agents. Some AMPs are also effective against protozoa or cancer cells. The tryptophan (Trp)-rich peptides (TRPs) are a subset of AMPs that display potent antimicrobial activity, credited to the unique biochemical properties of tryptophan that allow it to insert into biological membranes. Further, many Trp-rich AMPs cross bacterial membranes without compromising their integrity and act intracellularly, suggesting interactions with nucleic acids and enzymes. In this work, we overview some archetypal TRPs derived from natural sources, i.e., indolicidin, tritrpticin and lactoferricin, summarising their biochemical properties, structures, antimicrobial activities, mechanistic studies and potential applications.

Exposure of tryptophan (Trp) in aqueous solutions to UVA radiation resulted in decrease of Trp (C1) but generated an unknown fluorescent component (C2) with fluorescence emission maxima extending into wavelength range characteristic of humic substance (HS)-like material. The intensity of the two components (C1 and C2) could be operationally fit to linear functions of the illumination time t (0~40 h). However, C1 and C2 decreased and increased nonlinearly respectively in a mixture (Trp mixed with a reference sample of natural organic matter, i.e., NOM) which was exposed to the same UVA illumination, and the change of both C1 and C2 was faster than that in the absence of NOM. Moreover, the UV-Vis absorption maximum (ex = 278 nm) of Trp was faster removed for the mixture (after 5 h) than for Trp solutions without NOM (after 20 h). These observations suggested NOM-facilitated photobleaching of Trp and photoproduction of a new FDOM component under UVA illumination. Meanwhile, the fluorescence of the NOM in the absence of Trp was well represented by two HS-like components which decayed monotonically upon exposure to UVA light, while the photoinduced decay became nonmonotonic in the presence of Trp, and one component even increased with illumination during certain time window, indicating Trp-facilitated production of HS-like fluorescence signatures from NOM. The findings show that UVA-induced optical signature changes of tryptophan and HS-like materials are coupled and highlight the potential impact of absorption of solar UVA light by natural dissolved organic matter (DOM) on using the optical signatures to trace sources and sinks of DOM.

Free-radical scavenging by tryptophan and eight of its metabolites through electron transfer was investigated in aqueous solution at physiological pH, using density functional theory and the Marcus theory. A test set of 30 free radicals was employed. Thermochemical and kinetic data on the corresponding reactions are provided here for the first time. Two different pathways were found to be the most important: sequential proton loss electron transfer (SPLET) and sequential double proton loss electron transfer (SdPLET). Based on kinetic analyses, it is predicted that the tryptophan metabolites kynurenic acid and xanthurenic acid are the best free-radical scavengers among the tested compounds; they were estimated to be at least 24 and 12 times more efficient than Trolox for scavenging (•)OOH. These findings are in line with previous reports suggesting that the antioxidant activity that has been attributed to tryptophan is actually due to its metabolites, and they demonstrate the particular importance of phenolic metabolites to such activity. Graphical Abstract Kynurenic acid (KNA) and xanthurenic acid (XNA) are the major contributors to the free-radical scavenging activity of tryptophan.

We show that the interaction of aromatic amino acids with lipid bilayers can be characterized by conventional 1D [Formula: see text]H NMR spectroscopy using reference spectra obtained in isopropanol-d8/D[Formula: see text]O solutions. We demonstrate the utility of this method with three different peptides containing tyrosine, tryptophan, or phenylalanine amino acids in the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid membranes. In each case, we determine an equivalent isopropanol concentration (EIC) for each hydrogen site of aromatic groups, in essence constructing a map of the chemical environment. These EIC maps provide information on relative affinities of aromatic side chains for either PC or PS bilayers and also inform on amino acid orientation preference when bound to membranes.

A number of NMR methods possess the capability of probing chemical exchange dynamics in solution. However, certain drawbacks limit the applications of these NMR approaches, particularly, to a complex system. Here, we propose a procedure that integrates the regularized nonnegative least squares (NNLS) analysis of multiexponential T2 relaxation into Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments to probe chemical exchange in a multicompartmental system. The proposed procedure was validated through analysis of (19)F T2 relaxation data of 6-fluoro-DL-tryptophan in a two-compartment solution with and without bovine serum albumin. Given the regularized NNLS analysis of a T2 relaxation curve acquired, for example, at the CPMG frequency υ CPMG  = 125, the nature of two distinct peaks in the associated T2 distribution spectrum indicated 6-fluoro-DL-tryptophan either retaining the free state, with geometric mean */multiplicative standard deviation (MSD) = 1851.2 ms */1.51, or undergoing free/albumin-bound interconversion, with geometric mean */MSD = 236.8 ms */1.54, in the two-compartment system. Quantities of the individual tryptophan species were accurately reflected by the associated T2 peak areas, with an interconversion state-to-free state ratio of 0.45 ± 0.11. Furthermore, the CPMG relaxation dispersion analysis estimated the exchange rate between the free and albumin-bound states in this fluorinated tryptophan analog and the corresponding dissociation constant of the fluorinated tryptophan-albumin complex in the chemical-exchanging, two-compartment system.

Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins' unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.

UVR8 is a recently discovered ultraviolet-B (UV-B) photoreceptor protein identified in plants and algae. In the dark state, UVR8 exists as a homodimer, whereas UV-B irradiation induces UVR8 monomerization and initiation of signaling. Although the biological functions of UVR8 have been studied, the fundamental reaction mechanism and associated kinetics have not yet been fully elucidated. Here, we used the transient grating method to determine the reaction dynamics of UVR8 monomerization based on its diffusion coefficient. We found that the UVR8 photodissociation reaction proceeds in three stages: (i) photoexcitation of cross-dimer tryptophan (Trp) pyramids; (ii) an initial conformational change with a time constant of 50 ms; and (iii) dimer dissociation with a time constant of 200 ms. We identified W285 as the key Trp residue responsible for initiating this photoreaction. Although the C-terminus of UVR8 is essential for biological interactions and signaling via downstream components such as COP1, no obvious differences were detected between the photoreactions of wild-type UVR8 (amino acids 1-440) and a mutant lacking the C-terminus (amino acids 1-383). This similarity indicates that the conformational change associated with stage ii cannot primarily be attributed to this region. A UV-B-driven conformational change with a time constant of 50 ms was also detected in the monomeric mutants of UVR8. Dimer recovery following monomerization, as measured by circular dichroism spectroscopy, was decreased under oxygen-purged conditions, suggesting that redox reactivity is a key factor contributing to the UVR8 oligomeric state.

In the present work, we evaluated the role of gramicidin conformation in its photosensitized oxidation in organic solvents when irradiated in the presence of riboflavin. Gramicidin conformation has been described as monomeric in trifluoroethanol and as an intertwined dimer in methanol. Gramicidin showed extensive photo-oxidation upon irradiation in the presence of riboflavin in both solvents, and tryptophan residues were identified to be involved. We synthesized a gramicidin derivative methylated at position 1 of the indole ring of tryptophan to assess its effect on gramicidin conformation and photo-oxidation. Methylated gramicidin showed very similar absorption and emission spectra to gramicidin, but different conformations were identified by circular dichroism spectra. Upon irradiation, N-methylated tryptophan residues in the gramicidin derivative were not easily photo-oxidized by riboflavin compared to gramicidin. Circular dichroism spectra for gramicidin in methanol changed significantly upon irradiation in the presence of riboflavin indicating a change in conformation, while in trifluoroethanol no such changes were observed. Time-resolved fluorescence and anisotropy studies showed that oxidized gramicidin in methanol had shorter fluorescence lifetimes and a shorter rotational correlation time compared to non-irradiated gramicidin. Additionally, SDS-PAGE analysis showed a marked change in the electrophoretic pattern, whereas the high-molecular-weight bands disappeared upon irradiation. We interpret all these results in terms of a riboflavin photosensitized shift in gramicidin conformation from intertwined to monomeric.

Tryptophan residues at the dimer interface of the plant photoreceptor UVR8 promote monomerisation after UV-B absorption via a so far unknown mechanism. Using FTIR spectroscopy we assign light-induced structural transitions of UVR8 mainly to amino acid side chains without major transformations of the secondary structure of the physiologically relevant C-terminal extension. Additionally, we assign the monomerisation associated increase and red shift of the UVR8 tryptophan emission to a photoinduced rearrangement of tryptophan side chains and a relocation of the aspartic acid residues D96 and D107, respectively. By illumination dependent emission spectroscopy we furthermore determined the quantum yield of photoinduced monomerisation to 20 ± 8%.

Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.

A computational approach to designing a peptide-based ligand for the purification of human serum albumin (HSA) was undertaken using molecular docking and molecular dynamics (MD) simulation. A three-step procedure was performed to design a specific ligand for HSA. Based on the candidate pocket structure of HSA (warfarin binding site), a peptide library was built. These peptides were then docked into the pocket of HSA using the GOLD program. The GOLDscore values were used to determine the affinity of peptides for HSA. Consequently, the dipeptide Trp-Trp, which shows a high GOLDscore value, was selected and linked to a spacer arm of Lys[CO(CH2)5NH] on the surface of ECH-lysine sepharose 4 gel. For further evaluation, the Autodock Vina program was used to dock the linked compound into the pocket of HSA. The docking simulation was performed to obtain a first guess of the binding structure of the spacer-Trp-Trp-HSA complex and subsequently analyzed by MD simulations to assess the reliability of the docking results. These MD simulations indicated that the ligand-HSA complex remains stable, and water molecules can bridge between the ligand and the protein by hydrogen bonds. Finally, absorption spectroscopic studies were performed to illustrate the appropriateness of the binding affinity of the designed ligand toward HSA. These studies demonstrate that the designed dipeptide can bind preferentially to the warfarin binding site.

Iron-dependent enzymes are prevalent in nature and participate in a wide range of biological redox activities. Frequently, high-valence iron intermediates are involved in the catalytic events of iron-dependent enzymes, especially when the activation of peroxide or molecular oxygen is involved. Building on the fundamental framework of iron-oxygen chemistry, these reactive intermediates constantly attract significant attention from the enzymology community. During the past few decades, tremendous efforts from a number of laboratories have been dedicated to the capture and characterization of these intermediates to improve mechanistic understandings. In 2008, an unprecedented bis-Fe(IV) intermediate was reported in a c-type diheme enzyme, MauG, which is involved in the maturation of a tryptophan tryptophylquinone cofactor of methylamine dehydrogenase. This intermediate, although chemically equivalent to well-characterized high-valence iron intermediates, such as compound I, compound ES, and intermediate Q in methane monooxygenase, as well as the hypothetical Fe(V) species in Rieske non-heme oxygenases, is orders of magnitude more stable than these other high-valence species in the absence of its primary substrate. It has recently been discovered that the bis-Fe(IV) intermediate exhibits a unique near-IR absorption feature which has been attributed to a novel charge-resonance phenomenon. This review compares the properties of MauG with structurally related enzymes, summarizes the current knowledge of this new high-valence iron intermediate, including its chemical origin and structural basis, explores the formation and consequences of charge resonance, and recounts the long-range catalytic mechanism in which bis-Fe(IV) participates. Biological strategies for storing oxidizing equivalents with iron ions are also discussed.

Pin1 protein is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence etc., indicating an intermediate state around chromophores in Pin1 at about 4.0. Molecular dynamics simulations for the wild type Pin1 and its mutants were performed to explore the role of pH in the conformation changes of Pin1 protein. Our present study shows that one protein domain (PPIase domain) is more sensitive than the other one (WW domain) in Pin1 protein, and also our study shows that the integrality of the two conserve tryptophan in one domain (WW) is important in response to low pH. We arrive at the last result with the analysis of the protein root mean square distance and the analysis of the radius of gyration. The analysis of protein solvent accessible surface area values have proven our previous experiment result that there is an intermediate state around tryptophan residues at about pH 4.0. Moreover, acidic states of the protein can break the alpha-helixes in Pin1, especially the alpha-helix α3 close to active sites; as a result, Pin1 loses most of its activity at low pH. The results help us to understand the role of pH in Pin1, provide us insights into the conformation change at the atomic-level and emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for the related Pin1 diseases by targeting acidosis and modifying the intracellular pH gradients.

Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) maintains the cytoplasmic pH homeostasis of many bacteria and higher plants by coupling pyrophosphate (PPi) hydrolysis and proton translocation. H+-PPase accommodates several essential motifs involved in the catalytic mechanism, including the PPi binding motif and Acidic I and II motifs. In this study, 3 intrinsic tryptophan residues, Trp-75, Trp-365, and Trp-602, in H+-PPase from Clostridium tetani were used as internal probes to monitor the local conformational state of the periplasm domain, transmembrane region, and cytoplasmic domain, respectively. Upon binding of the substrate analog Mg-imidodiphosphate (Mg-IDP), local structural changes prevented the modification of tryptophan residues by N-bromosuccinimide (NBS), especially at Trp-602. Following Mg-Pi binding, Trp-75 and Trp-365, but not Trp-602, were slightly protected from structural modifications by NBS. These results reveal the conformation of H+-PPase is distinct in the presence of different ligands. Moreover, analyses of the Stern-Volmer relationship and steady-state fluorescence anisotropy also indicate that the local structure around Trp-602 is more exposed to solvent and varied under different environments. In addition, Trp-602 was identified to be a crucial residue in the H+-PPase that may potentially be involved in stabilizing the structure of the catalytic region by site-directed mutagenesis analysis.

Three reducing monosaccharides (glucose; Glc, galactose; Gal, and mannose; Man) were derivatized with L-tryptophan (L-Trp) under alkaline conditions. The DL-Gal and DL-Man derivatives were chirally resolved by HPLC with an acidic mobile phase, but the DL-Glc derivative was not. All of the three DL-monosaccharide derivatives were simultaneously enantioseparated using HPLC with a SunShell RP-AQUA column (C28) and a basic mobile phase. The optimum mobile phase conditions consisted of 5 mM phosphate and 25 mM tetraborate buffer (pH 9.6) at 20°C. With this system, resolution of D- and L-isomers of the Glc, Gal and Man derivatives were approximately 1.7, 2.2 and 2.4, respectively. When the three monosaccarides were derivatized with L-phenylalanine instead of L-Trp, DL-Gal and DL-Man were enantioseparated under both acidic and basic conditions, but DL-Glc was not. It was observed that enantiomer elution orders of the three monosaccharides derivatized with L-Trp were reasonably reversed when derivatized with D-Trp. It was also revealed that borate anions were required for simultaneous enantioseparation with HPLC.

ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and two-electrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.

The effect of reactive oxidation species (ROS) on tryptophan or tyrosine was investigated by qualitatively determining the major detectable oxidation products generated by hydroxyl radicals, produced by the Fenton process, or singlet oxygen, generated by exposure to green light in the presence of Rose Bengal, on these photosensitive amino acids in synthetic pentapeptides. Based on mass spectrometric analysis it would appear that the hydroxyl radical favours a pathway leading to the formation of tryptophandione-based products from tryptophan. In contrast singlet oxygen attack appears to favour the formation of kynurenine-type products from tryptophan. Specific oxidative products observed proteomically are therefore potentially able to discriminate between predominant ROS-mediated pathways. To validate these findings, a keratin-enriched extract was exposed to UVB light under aqueous conditions. The observation of the conversion of tryptophan to hydroxytryptophan in marker peptides, and the absence of singlet-oxygen specific modifications, suggested that under these conditions oxidative degradation occurred primarily via hydroxyl radical attack. These observations provide the first direct proteomic evidence of the dominant photodegradation pathways in wet wool.

β2-Microglobulin (β2m) is a small, monomorphic protein non-covalently bound to the heavy chain (HC) in polymorphic major histocompatibility complex (MHC) class I molecules. Given the high evolutionary conservation of structural features of β2m in various MHC molecules as shown by X-ray crystallography, β2m is often considered as a mere scaffolding protein. Using nuclear magnetic resonance (NMR) spectroscopy, we investigate here whether β2m residues at the interface to the HC exhibit changes depending on HC polymorphisms and the peptides bound to the complex in solution. First we show that human β2m can effectively be produced in deuterated form using high-cell-density-fermentation and we employ the NMR resonance assignments obtained for triple-labeled β2m bound to the HLA-B*27:09 HC to examine the β2m-HC interface. We then proceed to compare the resonances of β2m in two minimally distinct subtypes, HLA-B*27:09 and HLA-B*27:05, that are differentially associated with the spondyloarthropathy Ankylosing Spondylitis. Each of these subtypes is complexed with four distinct peptides for which structural information is already available. We find that only the resonances at the β2m-HC interface show a variation of their chemical shifts between the different complexes. This indicates the existence of an unexpected plasticity that enables β2m to accommodate changes that depend on HC polymorphism as well as on the bound peptide through subtle structural variations of the protein-protein interface.

Click chemistry was used as an efficient method to covalently attach a chromophore to an amino acid. Such easily prepared model systems allow for time-resolved studies of one-electron oxidation reactions by the excitation of the chromophore by a laser flash. The model complex ruthenium-tryptophan (Ru-Trp) has been synthesised and studied for its photophysical and electrochemical properties. Despite a small driving force of less than 100 meV, excitation with a laser flash results in fast internal electron transfer leading to the formation of the protonated radical (Trp˙H(+)). At neutral pH electron transfer is followed by deprotonation to form the neutral Trp˙ radical with the rate depending on the concentration of water acting as the proton acceptor. The formation of the tryptophan radical was confirmed by EPR.

Non-steroidal anti-inflammatory drugs (NSAID) are widely used in the treatment of pain and inflammation associated with several diseases. Naproxen, 2-(6-methoxy-2-naphthyl) propionic acid (NAP), belongs to this pharmacological class and appears to be associated with a high incidence of both photoallergic and phototoxic reactions. In this study, using human fibroblasts, we examined the biological effects of NAP photosensitization induced by UVA, the predominant UV component of sunlight reaching the Earth's surface. We showed that NAP or UVA alone have no cytotoxic effects at the concentrations and doses used in this study. The same result was observed when cells were pre-incubated with NAP but irradiated without NAP. In marked contrast, exposure of cells in the presence of NAP led to a drastic reduction of cell viability. These results suggest that the phototoxicity is mainly due to irradiation of extracellular NAP that damages cell membranes. Moreover, we showed that NAP itself led to a low but reproducible production of reactive oxygen species (ROS), to protein modifications by lipid peroxidation-derived aldehydes, to p38 phosphorylation and to the slowing-down of DNA replication, while UVA treatment alone showed no effects. NAP photosensitization with UVA led to protein S-glutathionylation, oxidation of the proliferating cell nuclear antigen (PCNA), oxidation of cellular tryptophan, phosphorylation of Chk1 and inhibition of DNA replication. However, using small interfering RNA to down regulate Chk1 expression in cells, we showed that Chk1 is not required to slow the S-phase down. Nevertheless, inhibition of Chk1, but not of p38, sensitized the cells to the phototoxic effects of NAP. Collectively, our data suggest that the interaction of NAP with the cells triggers oxidative damage and a replication stress, which are exacerbated by UVA radiation. As oxidative and replication stress-induced genome instability are important factors in aging and tumor predisposition, it is of interest to evaluate the consequence of a non-steroidal anti-inflammatory drug, like naproxen, on genomic instability.

Light-activation of photoactive yellow protein (PYP) is followed by a series of dynamical transitions in the structure of the protein. Tryptophan fluorescence is well-suited as a tool to study selected aspects of these. Using site-directed mutagenesis eight 'single-tryptophan' mutants of PYP were made by replacement of either a tyrosine, phenylalanine or histidine residue by tryptophan, while simultaneously eliminating the endogenous W119. Surprisingly, only three of these eight mutants turn out to emit measurable tryptophan fluorescence: F6W/W119F, F96W/W119F and H108W/W119F. Significantly, all three show altered tryptophan fluorescence upon formation of the pB state. As F96 is located very close to the chromophore, the F96W/W119F mutant protein is particularly suitable for further studies on the dynamical changes of the polarity in the chromophore-binding pocket of PYP. Furthermore, WT PYP can be photo-activated by a UV photon via the highly conserved W119 and subsequent Förster resonance energy transfer. Placing a unique tryptophan residue elsewhere in the protein shows that position 119 is favoured for UV-activation of PYP.