Stereoselectivity of Electron and Energy Transfer in the Quenching of (S/R)-Ketoprofen-(S)-Tryptophan Dyad Excited State

Photoinduced elementary processes in chiral linked systems, consisting of drugs and tryptophan (Trp) residues, attract considerable attention due to several aspects. First of all, these are models that allow one to trace the full and partial charge transfer underlying the binding of drugs to enzymes and receptors. On the other hand, Trp fluorescence is widely used to establish the structure and conformational mobility of proteins due to its high sensitivity to the microenvironment. Therefore, the study of mechanisms of Trp fluorescence quenching in various systems has both fundamental and practical interest. An analysis of the photo-chemically induced dynamic nuclear polarization (CIDNP) and Trp fluorescence quenching in (R/S)-ketoprofen-(S)-tryptophan ((S/R)-KP-(S)-Trp) dyad carried out in this work allowed us to trace the intramolecular reversible electron transfer (ET) and obtain evidence in favor of the resonance energy transfer (RET). The fraction of dyad’s singlet excited state, quenched via ET, was shown to be 7.5 times greater for the (S,S)-diastereomer than for the (R,S) analog. At the same time, the ratio of the fluorescence quantum yields shows that quenching effectiveness of (S,S)-diastereomer to be 5.4 times lower than for the (R,S) analog. It means that the main mechanism of Trp fluorescence quenching in (S/R)-KP-(S)-Trp dyad is RET.

Using submersible fluorescence sensors to track the removal of organic matter in decentralized wastewater treatment systems (DEWATS) in real time

Decentralized wastewater treatment systems (DEWATS) using anaerobic treatment are increasingly being considered for wastewater treatment with options for non-potable water reuse at the community scale. One challenge for ensuring performance and reliability of DEWATS is the lack of suitable on-site sensors to monitor failure or contamination events. In this study, the aim was to use in situ fluorescence sensors to track the performance of a DEWATS, consisting of an anaerobic baffled reactor (ABR) coupled to anaerobic filter (AF) and constructed wetland (CW) treatment processes. A submersible in situ fluorometer equipped with tryptophan (TRP) and chromophoric dissolved organic matter (CDOM) sensors was deployed in each chamber of the ABR-AF-CW system, and results showed that TRP fluorescence was preferentially removed over CDOM fluorescence throughout the system. Significant relationships between TRP fluorescence and chemical oxygen demand (COD) also suggested that TRP fluorescence could be used as a surrogate for COD and soluble COD concentrations. Strong agreement between results obtained from the 1D in situ fluorometer and those obtained from a 3D benchtop fluorometer lends further support to the use of in situ fluorescence sensors to track DEWATS performance.

Interaction of Small Zinc Complexes with Globular Proteins and Free Tryptophan

A series of eight water soluble anionic, cationic, and neutral zinc(II) complexes were synthesized and characterized. The interaction of these complexes with bovine serum albumin (BSA), human serum albumin (HSA), lysozyme, and free tryptophan (Trp) was investigated using steady-state fluorescence spectroscopy. Static and dynamic fluorescence quenching analysis based on Stern-Volmer kinetics was conducted, and the decrease in fluorescence intensity of the Trp residue(s) can be ascribed predominantly to static quenching that occurs when the Zn complex binds to the protein and forms a nonfluorescent complex. The role played by the nature of the ligand, the metal, and complex charge in quenching Trp fluorescence was investigated. The binding association constants (Ka) ranged from 104 to 1010 M−1 and indicate that complexes with planar aromatic features have the strongest affinity for globular proteins and free Trp. Complexes with nonaromatic features failed to interact with these proteins at or in the vicinity of the Trp residues. These interactions were studied over a range of temperatures, and binding was found to weaken with the increase in temperature and was exothermic with a negative change in entropy. The thermodynamic parameters suggest that binding of Zn complexes to the proteins is a highly spontaneous and favorable process.

Analysis of β3 Binding to the c-Src SH3 Domain

Abstract 383 An essential component of αIIbβ3-mediated outside-in signaling is activation of the tyrosine kinase c-Src, some of which is constitutively bound via its SH3 domain to the C-terminal Arg759-Gly760-Thr761 (RGT) sequence of the β3 cytoplasmic tail. RGT is quite different from the canonical polyproline sequence recognized by SH3 domains in which a polyproline helix packs against a shallow groove composed of aromatic residues (Tyr93, Tyr95, Tyr139 in c-Src). A specificity pocket located at the end of the groove and composed of residues from the n-Src- and RT-loops affects substrate specificity. Because of the obvious difference between RGT and polyproline sequences, we asked how RGT binds to the c-Src SH3 domain and what implications this has for c-Src regulation by αIIbβ3. Initially, we employed CD spectroscopy and tryptophan (Trp) fluorescence because these techniques are sensitive to changes in the local environment surrounding aromatic residues. However, there were no differences in the CD spectrum of the SH3 domain in absence or presence of the β3 peptide NITYRGT, whereas there was a clear shift in the presence of the core polyproline peptide RPLPPLP. Polyproline binding to Trp in the SH3 specificity pocket also results in a blue shift in Trp fluorescence from 355 nm to 347 nm; however, the fluorescence spectrum was essentially unchanged in the presence of NITYRGT. These experiments suggest that either the interaction of NITYRGT with SH3 is extremely weak and not observed at the concentrations used or occurs outside of the aromatic groove and the specificity pocket. Accordingly, we turned to NMR, a method able to detect weak protein-protein interactions. Two dimensional 1H-15N HSQC spectra of the SH3 domain in the presence of NITYRGT exhibited a number of changes in chemical shift compared to the spectrum in the absence of ligand. Sixteen residues located in the n-Src and RT-loops, grouped around the specificity pocket, had chemical shift changes > 0.05 ppm. The largest changes occurred in residues in or adjacent to the RT-loop, especially residues Arg98, Glu100, and Asp102. Of the resides forming the aromatic groove, only Tyr95 which is adjacent to the specificity pocket was perturbed by NITYRGT. Plots of the chemical shift changes for NH groups in SH3 vs. NITYRGT concentration were linear, indicating that the majority of SH3 domain was unbound. Further, a Kd for NITYRGT binding to SH3, estimated from these experiments, was between 175–350 mM. Next, we obtained HSQC spectra for SH3 in the presence of either RPLPPLP or a negative control peptide NITYEGK. Major perturbations due to RPLPPLP occurred in three regions: residues 98–103 (RT-loop), 116–122 (n-Src loop and specificity pocket), and residues 134–138; residues in the aromatic cluster were unaffected by the ligand. By contrast, only a handful of residues showed small perturbations in the presence of NITYEGK and there was no overlap between the affected residues and those affected by RPLPPLP. In conclusion, our results indicate that compared to polyproline sequences, the C-terminus of the β3 cytoplasmic tail binds to the c-Src SH3 domain in the region of the SH3 specificity pocket. Because chemical shifts for acidic residues located in the RT-loop were particularly sensitive to the presence of NITYRGT, it is likely that Arg759 in β3 makes an important contribution to the interaction. Moreover, we found that the interaction between NITYRGT and the c-Src SH3 domain is substantially weaker than was previously reported for the interaction of β3 with c-Src. This suggest the possibility that a third component is required for this interaction to occur under biological conditions. Recently we found that the β3 cytoplasmic tail in solution has weak affinity for the talin-1 FERM domain, but appending the tail to acidic phospholipids increased its affinity by three orders of magnitude. Since the c-Src SH3 domain contains a conserved patch of basic residues that are necessary for binding to acidic phospholipids, it is possible that the interaction of c-Src with β3 is also a ternary interaction in which protein-lipid interactions play an important role. Disclosures: No relevant conflicts of interest to declare.