IR Probes of Protein Microenvironments: Utility and Potential for Perturbation

A variety of IR‐active moieties with absorptions that are distinct from those of proteins have been developed as probes of local protein environments, including carbon‐deuterium bonds (C?D), cyano groups (CN), and azides (N₃); however, no systematic analysis of their utility in a protein has been published. Previously, we characterized the N‐terminal Src homology 3 domain of the murine adapter protein Crk‐II (nSH3) with C?D bonds site‐selectively incorporated throughout, and showed that it is relatively rigid and electrostatically heterogeneous and that it thermally unfolds under equilibrium conditions via a simple two‐state mechanism. We now report the synthesis and characterization of eight variants of nSH3 with CN and/or N₃ probes at five of the same positions. In agreement with previous studies, the position‐dependent spectra suggest that both probes are predominantly sensitive to hydration, and not to their local electrostatic environments. Importantly, both probes also tend to significantly perturb the protein if they are not incorporated at surface‐exposed positions. Thus, unlike C?D labels, which are both sensitive to their environment and non‐perturbative, CN and N₃ probes should be used with caution.     

Quenching of nucleotide-derived radicals by bis benzimidazole derivative Hoechst-33258 in aqueous solution

The pulse radiolysis technique has been employed to investigate the reaction of DNA-minor-groove ligand bisbenzimidazole Hoechst 33258 with pyrimidine and purine nucleotide-derived radicals. Formation of an N-centred Hoechst-33258 radical is observed. Bimolecular rate constants and the yields of Hoechst-33258 radical have been evaluated. While the rate constant for the reaction of pyrimidine-derived radicals with Hoechst-33258 remained the same (1–2) × 10⁹ dm³ mol⁻¹ s⁻¹, the yields of the Hoechst-33258 radical varied from 25% (5′-cytidine monophosphate) to 75% (5′-guanosine monophosphate) under anoxic conditions. The rate constant values for the reaction of purine-derived radicals with Hoechst-33258, under oxic and anoxic conditions, remained the same whereas with pyrimidine-derived radicals, the rate constant value under oxic conditions was about two orders of magnitude lower than under anoxic conditions. The difference in the yields of Hoechst-33258 radical with various nucleotide-derived radicals suggest the formation of different types of radicals and that the reaction mainly occurs by electron transfer from Hoechst-33258 to the nucleotide radicals.     

Statistical Physics Analysis of Maximum a Posteriori Estimation for Multi-channel Hidden Markov Models

The performance of Maximum a posteriori (MAP) estimation is studied analytically for binary symmetric multi-channel Hidden Markov processes. We reduce the estimation problem to a 1D Ising spin model and define order parameters that correspond to different characteristics of the MAP-estimated sequence. The solution to the MAP estimation problem has different operational regimes separated by first order phase transitions. The transition points for L-channel system with identical noise levels, are uniquely determined by L being odd or even, irrespective of the actual number of channels. We demonstrate that for lower noise intensities, the number of solutions is uniquely determined for odd L, whereas for even L there are exponentially many solutions. We also develop a semi analytical approach to calculate the estimation error without resorting to brute force simulations. Finally, we examine the tradeoff between a system with single low-noise channel and one with multiple noisy channels.     

Effect of base stacking on the acid-base properties of the adenine cation radical [A*+] in solution: ESR and DFT studies

In this study, the acid-base properties of the adenine cation radical are investigated by means of experiment and theory. Adenine cation radical (A*(+)) is produced by one-electron oxidation of dAdo and of the stacked DNA-oligomer (dA)6 by Cl2*(-) in aqueous glass (7.5 M LiCl in H2O and in D2O) and investigated by ESR spectroscopy. Theoretical calculations and deuterium substitution at C8-H and N6-H in dAdo aid in our assignments of structure. We find the pKa value of A*(+) in this system to be ca. 8 at 150 K in seeming contradiction to the accepted value of < or = 1 at ambient temperature. However, upon thermal annealing to > or = 160 K, complete deprotonation of A*(+) occurs in dAdo in these glassy systems even at pH ca. 3. A*(+) found in (dA)6 at 150 K also deprotonates on thermal annealing. The stability of A*(+) at 150 K in these systems is attributed to charge delocalization between stacked bases. Theoretical calculations at various levels (DFT B3LYP/6-31G*, MPWB95, and HF-MP2) predict binding energies for the adenine stacked dimer cation radical of 12 to 16 kcal/mol. Further DFT B3LYP/6-31G* calculations predict that, in aqueous solution, monomeric A*(+) should deprotonate spontaneously (a predicted pKa of ca. -0.3 for A*(+)). However, the charge resonance stabilized dimer AA*(+) is predicted to result in a significant barrier to deprotonation and a calculated pKa of ca. 7 for the AA*(+) dimer which is 7 pH units higher than the monomer. These theoretical and experimental results suggest that A*(+) isolated in solution and A*(+) in adenine stacks have highly differing acid-base properties resulting from the stabilization induced by hole delocalization within adenine stacks.     

Influence of chiral ionic liquids on the excited-state properties of naproxen analogs

The synthesis and decolorization of chiral room-temperature ionic liquids based upon 1-methyl imidazole and chloromethyl menthyl ether is reported. The excellent optical quality of these solvents permits the investigation of the effects of the two enantiomers on the excited-state photophysics of (S)-N-methyl-2-pyrrolidinemethyl 2(S)-(6-methoxy-2-naphthyl)propionate [(S,S)-NPX-PYR]. Whereas in conventional bulk polar solvents such as acetonitrile, (S,S)-NPX-PYR is known to execute excited-state intramolecular electron transfer and to form exciplexes, in these chiral solvents these nonradiative processes are absent. The chiral solvents do, however, induce a small but reproducible (approximately 10%) stereodifferentiation in the fluorescence lifetime of (S,S)-NPX-PYR as well as in the parent compound, (S)-naproxen. To our knowledge, this is the first example of chiral ionic liquids inducing such an effect on photophysical properties.     

Synthesis and optical characterization of c-axis oriented GaN thin films on amorphous quartz glass via sol–gel process

c-Axis oriented GaN nanocrystalline thin films were fabricated by nitridation of three different thin films of -GaO(OH), -Ga₂O₃ or β-Ga₂O₃ obtained by sol–gel technique on amorphous quartz glass substrates. All these GaN thin films showed near band edge emission at 390 nm and yellow luminescence at 570 nm. The crystalline nature and c-axis orientation as well as luminescence properties of the GaN thin films increased by several times by using a buffer layer of GaN on the substrate.     

K-P-Burgers equation in negative ion-rich relativistic dusty plasma including the effect of kinematic viscosity

The stationary solution is obtained for the K–P–Burgers equation that describes the nonlinear propagations of dust ion acoustic waves in a multi-component, collisionless, un-magnetized relativistic dusty plasma consisting of electrons, positive and negative ions in the presence of charged massive dust grains. Here, the Kadomtsev–Petviashvili(K–P) equation, threedimensional(3D) Burgers equation, and K–P–Burgers equations are derived by using the reductive perturbation method including the effects of viscosity of plasma fluid, thermal energy, ion density, and ion temperature on the structure of a dust ion acoustic shock wave(DIASW). The K–P equation predictes the existences of stationary small amplitude solitary wave,whereas the K–P–Burgers equation in the weakly relativistic regime describes the evolution of shock-like structures in such a multi-ion dusty plasma.     

Prototropic equilibria in DNA containing one-electron oxidized GC: intra-duplex vs. duplex to solvent deprotonation

By use of ESR and UV-vis spectral studies, this work identifies the protonation states of one-electron oxidized G:C (viz. G˙+:C, G(N1–H)˙:C(+H+), G(N1–H)˙:C, and G(N2-H)˙:C) in a DNA oligomer d[TGCGCGCA]2. Benchmark ESR and UV-vis spectra from one electron oxidized 1-Me-dGuo are employed to analyze the spectral data obtained in one-electron oxidized d[TGCGCGCA]2 at various pHs. At pH ≥7, the initial site of deprotonation of one-electron oxidized d[TGCGCGCA]2 to the surrounding solvent is found to be at N1 forming G(N1–H)˙:C at 155 K. However, upon annealing to 175 K, the site of deprotonation to the solvent shifts to an equilibrium mixture of G(N1–H)˙:C and G(N2–H)˙:C. For the first time, the presence of G(N2–H)˙:C in a ds DNA-oligomer is shown to be easily distinguished from the other prototropic forms, owing to its readily observable nitrogen hyperfine coupling (Azz(N2) = 16 G). In addition, for the oligomer in H2O, an additional 8 G N2–H proton HFCC is found. This ESR identification is supported by a UV-vis absorption at 630 nm which is characteristic for G(N2–H)˙ in model compounds and oligomers. We find that the extent of photo-conversion to the C1′ sugar radical (C1′˙) in the one-electron oxidized d[TGCGCGCA]2 allows for a clear distinction among the various G:C protonation states which can not be easily distinguished by ESR or UV-vis spectroscopies with this order for the extent of photo-conversion: G˙+:C > G(N1–H)˙:C(+H+) ? G(N1–H)˙:C. We propose that it is the G˙+:C form that undergoes deprotonation at the sugar and this requires reprotonation of G within the lifetime of exited state