Walter G. Gonzalez

Graphene Oxide as a Quencher for Fluorescent Assay of Amino Acids, Peptides, and Proteins

Understanding the interaction between graphene oxide (GO) and the biomolecules is fundamentally essential, especially for disease- and drug-related peptides and proteins. In this study, GO was found to strongly interact with amino acids (tryptophan and tyrosine), peptides (Alzheimers disease related amyloid beta 1-40 and type 2 diabetes related human islet amyloid polypeptide), and proteins (drug-related bovine and human serum albumin) by fluorescence quenching, indicating GO was a universal quencher for tryptophan or tyrosine related peptides and proteins. The quenching mechanism between GO and tryptophan (Trp) or tyrosine (Tyr) was determined as mainly static quenching, combined with dynamic quenching (Förster resonance energy transfer). Different quenching efficiency between GO and Trp or Tyr at different pHs indicated the importance of electrostatic interaction during quenching. Hydrophobic interaction also participated in quenching, which was proved by the presence of nonionic amphiphilic copolymer Pluronic F127 (PF127) in GO dispersion. The strong hydrophobic interaction between GO and PF127 efficiently blocked the hydrophobic interaction between GO and Trp or Tyr, lowering the quenching efficiency.

Flavin adenine dinucleotide structural motifs: from solution to gas phase.

Flavin adenine dinucleotide (FAD) is involved in important metabolic reactions where the biological function is intrinsically related to changes in conformation. In the present work, FAD conformational changes were studied in solution and in gas phase by measuring the fluorescence decay time and ion-neutral collision cross sections (CCS, in a trapped ion mobility spectrometer, TIMS) as a function of the solvent conditions (i.e., organic content) and gas-phase collisional partner (i.e., N2 doped with organic molecules). Changes in the fluorescence decay suggest that FAD can exist in four conformations in solution, where the abundance of the extended conformations increases with the organic content. TIMS-MS experiments showed that FAD can exist in the gas phase as deprotonated (M = C27H31N9O15P2) and protonated forms (M = C27H33N9O15P2) and that multiple conformations (up to 12) can be observed as a function of the starting solution for the [M + H]+ and [M + Na]+molecular ions. In addition, changes in the relative abundances of the gas-phase structures were observed from a “stack” to a “close” conformation when organic molecules were introduced in the TIMS cell as collision partners. Candidate structures optimized at the DFT/B3LYP/6-31G(d,p) were proposed for each IMS band, and results showed that the most abundant IMS band corresponds to the most stable candidate structure. Solution and gas-phase experiments suggest that the driving force that stabilizes the different conformations is based on the interaction of the adenine and isoalloxazine rings that can be tailored by the “solvation” effect created with the organic molecules.

Emission Tuning of Fluorescent Kinase Inhibitors: Conjugation Length and Substituent Effects

Fluorescent N-phenyl-4-aminoquinazoline probes targeting the ATP-binding pocket of the ERBB family of receptor tyrosine kinases are reported. Extension of the aromatic quinazoline core with fluorophore “arms” through substitution at the 6- position of the quinazoline core with phenyl, styryl, and phenylbutadienyl moieties was predicted by means of TD-DFT calculations to produce probes with tunable photoexcitation energies and excited states possessing charge-transfer character. Optical spectroscopy identified several synthesized probes that are nonemissive in aqueous solutions and exhibit emission enhancements in solvents of low polarity, suggesting good performance as turn-on fluorophores. Ligand-induced ERBB2 phosphorylation assays demonstrate that despite chemical modification to the quinazoline core these probes still function as ERBB2 inhibitors in MCF7 cells. Two probes were found to exhibit ERBB2-induced fluorescence, demonstrating the utility of these probes as turn-on, fluoroescent kinase inhibitors.

Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM.

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, Kv4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca(2+)DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS-DREAM complexes increases upon Ca(2+) association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca(2+)DREAM were determined to be 195±20μM and 62±4μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of Kv4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.

Modulation of the Voltage-gated Potassium Channel (Kv4.3) and the Auxiliary Protein (KChIP3) Interactions by the Current Activator NS5806

Background: KChIP3 association with Kv4 channels regulate the K+ current gating. Results: NS5806 binds to KChIP3 and stabilizes the KChIP3-Kv4 complex. Conclusion: Ca2+ or NS5806 binding on KChIP3 decreases the dissociation rate of Kv4.3. Significance: The role of the hydrophobic cavity on KChIP3 in drug and protein association could lead to a better drug design for treatment of heart conditions. KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2–5 μm in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μm and 2.7 ± 0.1 μm in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (Kd = 1.9 ± 0.1 μm) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of KV4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.

Emission switching of 4,6-diphenylpyrimidones: solvent and solid state effects.

The photophysics of 1-ethyl-4,6-bis(4-methoxyphenyl)-2(1H)-pyrimidone (1) and 1-ethyl-4,6-bis(4-(dimethylamino)phenyl)-2(1H)-pyrimidone (2) were investigated to determine the mechanisms of emission switching in response to protonation. UV-vis and steady state emission spectroscopy of the protonated and unprotonated forms across a range of solvents reveal the polarity dependence of the vertical excitation energies. Emission lifetimes and quantum yields show the solvent dependency of the excited states. Emission enhancements were observed in polyethylene glycol solutions and in the solid state (both thin film and single crystal), demonstrating the role of intramolecular rotation in thermal relaxation of the excited states. TD-DFT calculations provide insights into the excited state geometries and the role of intramolecular charge transfer. The collected data show that emission of diphenylpyrimidones can be modulated by four factors, including the identity of the electron-donating auxochrome, protonation state, solvent polarity, and viscosity.

Photophysical Characterization of a Benzo-Fused Analogue of Brooker’s Merocyanine: Solvent Polarity and pH Effects

The photophysical properties of 4-[2-(6-hydroxy-2-naphthalenyl)-ethenyl]-1-methyl-pyridinium (HNEP(+)) and its deprotonated form (NEP), a benzofused derivative of Brookers merocyanine (BM), were investigated through a combined spectroscopic and computational approach. Despite their structural similarities and similar pK(a) values, HNEP(+)/NEP and BMH(+)/BM differ in the extent of charge delocalization in the ground and excited states. NEP exhibits the spectral characteristics of a charge transfer species in solvents in which BM exists in a charge-delocalized quinoid; however, quantum chemical calculations show that the CT absorption of NEP is not necessarily a consequence of the zwitterionic character. HNEP(+) displays larger Stokes shifts than BMH(+), and NEP demonstrates enhanced solvatochromism relative to BM as a consequence of benzofusion.

Characterization of the Photophysical, Thermodynamic and Structural Properties of the Terbium(III)-KChIP3 Complex

DREAM (also known as K(+) channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca(2+)-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimers disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb(3+), are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb(3+). We also observe that unlike Tb(3+)-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τaverage ∼ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb(3+) binds to at least three sites with high affinity (Kd = 1.8 μM in the presence of Ca(2+)) and displaces bound Ca(2+) through an entropically driven mechanism (ΔH ∼ 12 kcal mol(-1), and TΔS ∼ 22 kcal mol(-1)). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb(3+), like Ca(2+), triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca(2+) binding, Tb(3+) binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry-mass spectrometry reveal that replacement of Ca(2+) with Tb(3+) preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM-protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.

Synthesis of 8-(1,2,3-triazol-1-yl)-7-deazapurine nucleosides by azide–alkyne click reactions and direct CH bond functionalization

Treatment of toyocamycin or sangivamycin with 1,3-dibromo-5,5-dimethylhydantoin in MeOH (r.t./30 min) gave 8-bromotoyocamycin and 8-bromosangivamycin in good yields. Nucleophilic aromatic substitution of 8-bromotoyocamycin with sodium azide provided novel 8-azidotoyocamycin. Strain promoted click reactions of the latter with cyclooctynes resulted in the formation of the 1,2,3-triazole products. Iodine-mediated direct C8-H bond functionalization of tubercidin with benzotriazoles in the presence of tert-butyl hydroperoxide gave the corresponding 8-benzotriazolyltubercidin derivatives. The 8-(1,2,3-triazol-1-yl)-7-deazapurine derivatives showed moderate quantum yields and a large Stokes shifts of ~ 100 nm.

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator.

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca2+ concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo‐state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca2+/Mg2+ association to DREAM. Our data indicate a minor impact of Ca2+ association on the overall structure of the N‐ and C‐terminal domains, although Ca2+ binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca2+ bound forms of the protein. Time‐resolved fluorescence data indicate that Ca2+binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca2+ association to EF‐hand 3 and EF‐hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca2+ and Mg2+ suggesting that, under physiological conditions, Ca2+ free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.