pH dependence of charge transfer between tryptophan and tyrosine in dipeptides

Time-resolved absorption spectroscopy has been employed to study the directionality and rate of charge transfer in W-Y and Ac-W-Y dipeptides as a function of pH. Excitation with 266-nm nanosecond laser pulses produces both W⋅ (or [⋅WH]⁺, depending on pH) and Y⋅. Between pH 6 and 10, W⋅ to was found to oxidize Y with k_X⋅=9.0×10⁴ s⁻¹ and 1.8×10⁴ s⁻¹ for the W-Y and Ac-W-Y dipeptide systems, respectively. The intramolecular charge transfer rate increases as the pH is lowered over the range 6>pH>2. For 10<pH<12, the rate of radical transport for the W-Y dipeptide decreases and becomes convoluted with other radical decay processes, the timescales of which have been identified in studies of control dipeptides Ac-F-Y and W-F. Further increases in pH prompt the reverse reaction to occur, W-Y⋅→W⋅-Y⁻ (Y⁻, tyrosinate anion), with a rate constant of k_X⋅=1.2×10⁵ s⁻¹. The dependence of charge transfer directionality between W and Y on pH is important to the enzymatic function of several model and natural biological systems as discussed here for ribonucleotide reductase.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

大肠杆菌trpBA和serA基因的串联表达

大肠杆菌trpBA基因编码的色氨酸合成酶(tryptophan synthetase, TSase)是色氨酸合成的关键酶; serA基因编码的磷酸甘油酸脱氢酶(D-3-phosphoglycerate-dehydrogenase, PGDH)为L-丝氨酸合成(色氨酸合成的底物)的关键酶。为了通过基因工程手段来增加色氨酸的产量, 在利用高效的原核表达载体pET22b(+)分别对trpBA和serA基因克隆表达的基础上, 采用PCR方法扩增了抗反馈抑制的serA和trpBA基因, 将两基因串联于pET22b(+)载体上, 共构建了4种方式的串联质粒, 实现了2种蛋白酶在大肠杆菌中的共表达。聚丙烯酰胺电泳分析显示, ABA-Ⅰ重组菌株在37 kD (PGDH)、29 kD(色氨酸合成酶的α亚基)、44 kD(β亚基)处均有明显的蛋白表达带。4种串联表达质粒重组菌的TSase酶活性, 分别比含空载体菌相应酶的活性提高2~4倍, PGDH酶活性分别提高约2.1~3.6倍。经摇瓶发酵实验表明酶活性较高的ABA-I菌株色氨酸合成量亦最高, 约为对照菌株的20.2倍。     

Effect of indoleamine dioxygenase-1 deficiency and kynurenine pathway inhibition on murine cerebral malaria

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. In this study, two different approaches were used to examine the role of indoleamine 2,3-dioxygenase-1 (IDO-1) and its metabolites in the development of murine CM. Mice genetically deficient in IDO-1 were not protected against CM, but partial protection was observed in C57BL/6 mice treated with Ro 61-8048, an inhibitor of kynurenine-3-hydroxylase. This protection was associated with suppressed levels of picolinic acid (PA) within the brain, but not with changes in the levels of kynurenic acid (KA) or quinolinic acid (QA). These data suggest that although IDO-1 is not directly involved in the pathogenesis of CM in C57BL/6 mice, the production of the kynurenine pathway metabolite PA may contribute to the development of murine CM.     

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects

Tryptophan indole-lyase (Trpase), PBPRA2532, from Photobacterium profundum SS9, a piezophilic marine bacterium, has been cloned, expressed in Escherichia coli, and purified. The P. profundum Trpase (PpTrpase) exhibits similar substrate specificity as the enzyme from E. coli (EcTrpase). PpTrpase has an optimum temperature for activity at about 30 °C, compared with 53 °C for EcTrpase, and loses activity rapidly (t_1/2 ∼ 30 min) when incubated at 50 °C, while EcTrpase is stable up to 65 °C. PpTrpase retains complete activity when incubated more than 3 h at 0 °C, while EcTrpase has only about 20% remaining activity. Under hydrostatic pressure, PpTrpase remains fully active up to 100 MPa (986 atm), while EcTrpase exhibits only about 10% activity at 100 MPa. PpTrpase forms external aldimine and quinonoid intermediates in stopped-flow experiments with l-Trp, S-Et-l-Cys, S-benzyl-l-Cys, oxindolyl-l-Ala, l-Ala and l-Met, similar to EcTrpase. However, with l-Trp a gem-diamine is observed that decays to a quinonoid complex. An aminoacrylate is observed with l-Trp in the presence of benzimidazole, as was seen previously with EcTrpase [28] but not with S-Et-l-Cys. The results show that PpTrpase is adapted for optimal activity in the low temperature, high pressure marine environment.     

Changes in PLA(2) activity after interacting with anti-inflammatory drugs and model membranes: evidence for the involvement of tryptophan residues

Phospholipase A₂ (PLA₂) lipolytic activity can be regarded as a limiting factor for the development of inflammatory processes by restricting the production of pro-inflammatory mediators, hence representing a valuable therapeutic target for drugs that are able to modulate the activity of this enzyme. In the current work, the hydrolysis of phospholipids by PLA₂ was monitored with acrylodan-labelled intestinal fatty acid binding protein (ADIFAB) and this fluorescence based technique was also used to access the enzymatic inhibitory effect of non-steroidal anti-inflammatory drugs (NSAIDs). The intrinsic fluorescence of PLA₂ tryptophan residues was further used to gain complementary information regarding the accessibility of these residues on the PLA₂ structure upon interaction with the NSAIDs tested; and to calculate the NSAIDs-PLA₂ binding constants. Finally, circular dichroism (CD) measurements were performed to evaluate changes in PLA₂ conformation resultant from the inhibitory effect of the drugs tested. Overall, results gathered in this study point to the conclusion that the studied NSAIDs inhibit PLA₂ activity due to a disturbance of the enzyme binding efficiency to membrane interface possibly by a shielding effect of the Trp residues required for the membrane interfacial binding step that precedes lipolysis process.     

Pea lectin unfolding reveals a unique molten globule fragment chain

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains -a long β-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a β-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ∼ 20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule β-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.     

The N-terminal Domain Tethers the Voltage-gated Calcium Channel β2e-subunit to the Plasma Membrane via Electrostatic and Hydrophobic Interactions

The β-subunit associates with the α₁ pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β_2a and β_2e, associate with the plasma membrane in the absence of the α₁-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β_2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β_2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β_2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β_1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type β_2e, but not with a β_2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β_2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the β-subunit family that also sustains slowed inactivation.     

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

Phytochromes are photoreceptors using a bilin tetrapyrrole as chromophore, which switch in canonical phytochromes between red (P_r) and far red (P_fr) light-absorbing states. Cph2 from Synechocystis sp., a noncanonical phytochrome, harbors besides a cyanobacteriochrome domain a second photosensory module, a P_r/P_fr-interconverting GAF-GAF bidomain (SynCph2(1-2)). As in the canonical phytochromes, a unique motif of the second GAF domain, the tongue region, seals the bilin-binding site in the GAF1 domain from solvent access. Time-resolved spectroscopy of the SynCph2(1-2) module shows four intermediates during P_r → P_fr phototransformation and three intermediates during P_fr → P_r back-conversion. A mutation in the tongue''s conserved PRXSF motif, S385A, affects the formation of late intermediate R3 and of a P_fr-like state but not the back-conversion to P_r via a lumi-F-like state. In contrast, a mutation in the likewise conserved WXE motif, W389A, changes the photocycle at intermediate R2 and causes an alternative red light-adapted state. Here, back-conversion to P_r proceeds via intermediates differing from SynCph2(1-2). Replacement of this tryptophan that is ∼15 Å distant from the chromophore by another aromatic amino acid, W389F, restores native P_r → P_fr phototransformation. These results indicate large scale conformational changes within the tongue region of GAF2 during the final processes of phototransformation. We propose that in early intermediates only the chromophore and its nearest surroundings are altered, whereas late changes during R2 formation depend on the distant WXE motifs of the tongue region. Ser-385 within the PRXSF motif affects only late intermediate R3, when refolding of the tongue and docking to the GAF1 domain are almost completed.