Modification of the Photochemical Reactions of Protein Tryptophanyls by Chloroform

The modifying action of chloroform and other halogenmethanes (difluorodichloromethane and carbon tetrachloride) on UV radiation-induced photochemical reactions of the protein tryptophanyls is shown. The effect manifests itself in an increase in the quantum yield of indole chromophore destruction and in formation of new photochemical products. It is found that the nature of the products of photochemical destruction which are formed in a tryptophan solution and in proteins is the same.     

Chemical Evolution of a Bacterial Proteome

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L ‐β‐(thieno[3,2‐b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L ‐tryptophan (Trp). A long‐term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20 899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new‐to‐nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.     

Total Synthesis of cis‐Clavicipitic Acid from Asparagine via Ir‐Catalyzed CH bond Activation as a Key Step

4‐Substituted tryptophan derivatives and the total synthesis of cis‐clavicipitic acid were achieved in reactions in which Ir‐catalyzed C?H bond activation was a key step. The starting material for these reactions is asparagine, which is a cheap natural amino acid. The reductive amination step from the 4‐substituted tryptophan derivative gave cis‐clavicipitic acid with perfect diastereoselectivity.     

壳聚糖/多壁碳纳米管复合膜电化学手性识别色氨酸对映异构体

采用滴涂方式将羧酸化多壁碳纳米管(f-MWCNTs)修饰于玻碳电极(GCE)表面成膜,然后恒电位法在上述修饰电极表面电沉积壳聚糖(CS)膜,形成CS和f-MWCNTs复合膜修饰电极(CS/f-MWCNTs/GCE),并用于色氨酸(Trp)对映异构体的手性识别。采用扫描电子显微镜(SEM)表征了修饰电极表面形貌的差异,电化学阻抗(EIS)和循环伏安法(CV)研究修饰电极的电化学行为差异。差分脉冲伏安法(DPV)用于区别色氨酸(Trp)对映异构体,分离系数可达2.38。研究发现该修饰电极对L-Trp的DPV响应信号强于D-Trp,检测的线性范围为8.0×10~(-6)~4.0×10~(-3)mol/L,检出限(S/N=3)为5.0×10~(-6)mol/L。该方法简单、经济、快速,对发展其它手性化合物的检测方法提供了参考。     

Mass spectrometric characterization of peptides containing different oxidized tryptophan residues

The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.     

Unexpected electron transfer in cryptochrome identified by time-resolved EPR spectroscopy

Subtle differences in the local sequence and conformation of amino acids can result in diversity and specificity in electron transfer (ET) in proteins, despite structural conservation of the redox partners. For individual ET steps, distance is not necessarily the decisive parameter; orientation and solvent accessibility of the ET partners, and thus the stabilization of the charge-separated states, contribute substantially.     

Time-resolved tryptophan fluorescence in flavodoxins

The time-resolved fluorescence characteristics of tryptophan in flavodoxins isolated from the bacteriaDesulfovibrio gigas, Desulfovibrio vulgaris, Clostridium beijerinckii, andMegasphaera elsdenii were examined. The fluorescence decays were recorded using pulsed synchrotron radiation as the excitation source and time-correlated single-photon counting in detection. The results were analyzed as lifetime distributions using the maximum entropy method. Comparison of the fluorescence decays of normal and flavin mononucleotide-depleted flavodoxins demonstrates that radiationless energy transfer from tryptophan to flavin occurs in all flavodoxins investigated. On comparing the lifetime distribution patterns of apo and holoflavodoxins, it was noticed that a certain amount of apoprotein is present in all holoflavodoxin samples. The three-dimensional structure of two flavodoxins allowed us to compare experimental with theoretical transfer rates and the results were in fair agreement.     

Facile synthesis of a cis-syn thymine dimer building block and its incorporation into oligodeoxynucleotides

The synthesis of a building block containing the photobiologically relevant cis-syn thymine cyclobutane photoproduct and its incorporation into oligonucleotides by the phosphoramidite-based solid-phase synthesis is reported. Compared to previous syntheses, this route is extremely short and allows such modified oligonucleotides to be easily available for biological studies.     

Triplet state sublevel spectroscopy applied to proteins

Triplet state sublevel spectroscopy using optical detection of magnetic resonance (ODMR) in zero magnetic field can be successfully employed to study (i) the environment of tryptophan (Trp) residues in a protein by observing the position and structure of phosphorescence spectra, zero field ODMR transitions and triplet state sublevel kinetics, (ii) the energy transfer among Trp residues, and (iii) whether any cysteine (Cys) residue is within van der Waals distance of any Trp residue by studying the complex of the protein with methylmercury(II) iodide (CH₃HgI) which binds to Cys residues. These studies are particularly important where crystal structure study is not possible. Study of the S₁ state often gives ambiguous results since fluorescence is always broad and shows multi-exponential decay. Our results on bacteriophage lysozyme T4 which contains three Trp residues at positions 126, 138 and 158 are presented. Measurements were facilitated by the use of a mutated enzyme containing one or two Trp-Tyr substitutions. The results indicate that (i) Trp 126 and 158 are solvent exposed, whereas Trp 138 is buried in a hydrophobic environment, (ii)S ↔S non-radiative energy transfer takes place predominantly from Trp 126 to Trp 158, and (iii) only Trp-158 undergoes a heavy atom perturbation, which affects selectively the z-sublevel (z is an out-of-plane axis of the indole plane) as a result of CH₃HgI binding to nearby Cys 97. We suggest that the Hg atom is located on the z-axis of Trp 158 in the complex. This interpretation is based on our investigations on the effect of orientation of heavy atom perturbers in some naphthalene-crown ether metal ion complexes.