PET and FRET utility of an amino acid pair: tryptophan and 4-cyanotryptophan.

Methods based on fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) are widely used in the biological sciences, employing mostly dye-based FRET and PET pairs. While very useful and important, dye-based reporters are not always applicable without concern, for example, in cases where the fluorophore size needs to be minimized. Therefore, development and characterization of smaller, ideally amino acid-based PET and FRET pairs will expand the biological spectroscopy toolbox to enable new applications. Herein, we show that, depending on the excitation wavelength, tryptophan and 4-cyanotrptophan can interact with each other via the mechanism of either energy or electron transfer, hence constituting a dual FRET and PET pair. The biological utility of this amino acid pair is further demonstrated by applying it to study the end-to-end collision rate of a short peptide, the mode of interaction between a ligand and BSA, and the activity of a protease.

4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA⁻protein interactions.

Newfound effect of N-acetylaspartate in preventing and reversing aggregation of amyloid-beta in vitro.

Although N-acetylaspartate (NAA) has long been recognized as the most abundant amino acid in neurons by far, its primary role has remained a mystery. Based on its unique tertiary structure, we explored the potential of NAA to modulate aggregation of amyloid-beta (Aß) peptide 1-42 via multiple corroborating aggregation assays along with electron microscopy. Thioflavin-T fluorescence assay demonstrated that at physiological concentrations, NAA substantially inhibited the initiation of Aß fibril formation. In addition, NAA added after 25 min of Aß aggregation was shown to break up preformed fibrils. Electron microscopy analysis confirmed the absence of mature fibrils following NAA treatment. Furthermore, fluorescence correlation spectroscopy and dynamic light scattering measurements confirmed significant reductions in Aß fibril hydrodynamic radius following treatment with NAA. These results suggest that physiological levels of NAA could play an important role in controlling Aß aggregation in vivo where they are both found in the same neuronal compartments.

Fermi resonance as a means to determine the hydrogen-bonding status of two infrared probes.

The C[double bond, length as m-dash]O/C[triple bond, length as m-dash]N stretching vibration arising from a carbonyl/nitrile functional group in various molecular systems has been frequently used to assess, for example, local hydrogen-bonding interactions, among other applications. However, in practice it is not always easy to ascertain whether the carbonyl or nitrile group in question is engaged in such interactions. Herein, we use 4-cyanoindole and cyclopentanone as models to show that, when a fundamental C[double bond, length as m-dash]O or C[triple bond, length as m-dash]N stretching mode is involved in Fermi resonance, the underlying vibrational coupling constant (W) is a convenient reporter of the hydrogen-bonding status of the corresponding carbonyl or nitrile group. Specifically, we find that for both groups a W value of 7.7 cm-1 or greater is indicative of their involvement in hydrogen-bonding interactions. Furthermore, we find that, as observed in similar studies, the Fermi resonance coupling leads to quantum beats in the two-dimensional infrared spectra of 4-cyanoindole in isopropanol, with a period of about 1.9 ps.

Kinetic Isotope Effect Provides Insight into the Vibrational Relaxation Mechanism of Aromatic Molecules: Application to Cyano-phenylalanine.

Varying the reduced mass of an oscillator via isotopic substitution provides a convenient means to alter its vibrational frequency and hence has found wide applications. Herein, we show that this method can also help delineate the vibrational relaxation mechanism, using four isotopomers of the unnatural amino acid p-cyano-phenylalanine (Phe-CN) as models. In water, the nitrile stretching frequencies of these isotopomers, Phe-(12)C(14)N (1), Phe-(12)C(15)N (2), Phe-(13)C(14)N (3), and Phe-(13)C(15)N (4), are found to be equally separated by ∼27 cm(-1), whereas their vibrational lifetimes are determined to be 4.0 ± 0.2 (1), 2.2 ± 0.1 (2), 3.4 ± 0.2 (3), and 7.9 ± 0.5 ps (4), respectively. We find that an empirical relationship that considers the effective reduced mass of CN can accurately account for the observed frequency gaps, while the vibrational lifetime distribution, which suggests an intramolecular relaxation mechanism, can be rationalized by the order-specific density of states near the CN stretching frequency.

Matrix and polymer soft-landing isolation of selected acids with pyridine and poly(4-vinylpyridine): a comparative infrared spectroscopic study of hydrogen bonding.

Hydrogen bonding plays a key role in the formation of nanostructures, as it is the "glue" between layers that are built by the layer-by-layer technique. Poly(4-vinylpyridine) (PVP) is one of the most commonly used polymers in these sandwich-structured films, often in conjunction with poly(carboxylic acid)s such as poly(acrylic acid) in the PVP/PAA interpolymer complex. In addition, PVP is commonly used as a polymer matrix for embedding semiconductor nanoparticles. In this study, hydrogen-bonded complexes of water, formic acid, and pentachlorocyclopropane, with pyridine in a traditional matrix isolation experiment and PVP in a novel "polymer soft-landing" isolation experiment, have been characterized for the first time at 16 K. Changes in vibrational modes of the proton donor species and in some cases pyridine modes provided ample evidence for complex formation. In the case of water and pentachlorocyclopropane, the matrix and polymer soft-landing results were quite similar, whereas formic acid formed a significantly different complex with pyridine in the argon matrix than with the pyridine ring on the PVP polymer. This work demonstrates clearly the benefit of using both the conventional matrix isolation technique and our polymer soft-landing variation in tandem to probe the structure of these complexes and thus elucidate the nature of the C-H···N, C-H···O═C, and O-H···N linkages.