We demonstrate here that a nitrile-derivatized phenylalanine residue, p-cyanophenylalanine (Phe(CN)), and tryptophan (Trp) constitute a novel donor-acceptor pair for fluorescence resonance energy transfer (FRET). The Förster distance of this FRET pair was determined to be approximately 16 A and hence is well suited for determining relatively short separation distances. To validate the applicability of this FRET pair in conformational studies, we studied the conformational heterogeneity of a 14-residue amphipathic peptide, Mastoparan X (MPx peptide), in water and 7 M urea solution as well as at different temperatures. Specifically, seven nitrile-derivatized mutants of the MPx peptide, each containing a Phe(CN) residue that replaces different positions along the peptide sequence (i.e., from position 5 to 11) and serves as a resonance energy donor to the native Trp residue at position 3, were studied spectroscopically. The FRET efficiencies obtained from these peptides allowed us to gain a global picture regarding the conformational distribution of the MPx peptide in different environments. Our results suggest that the MPx molecules exist in water as an ensemble of rather compact conformations, with a radius of gyration of approximately 4.2 A, whereas in 7 M urea the radius of gyration increases to approximately 6.5 A, indicating that the peptide conformations become more extended under this condition. However, we found that temperature had only a negligible effect on the size of the MPx peptide, underlining the difference between the thermally and chemically denatured states of polypeptides. The application of the Gaussian chain or the wormlike chain model allowed us to further obtain the probability distribution function of the separation distance between any two residues along the peptide sequence. We found that the effective bond length of the MPx peptide, obtained by using the Gaussian chain model, is 2.78 A in water and 4.28 A in 7 M urea.

The thermal stability and folding kinetics of a 15-residue beta-hairpin (SESYINPDGTWTVTE) have been studied by using infrared (IR) spectroscopy coupled with laser-induced temperature-jump (T-jump) technique for rapid folding-unfolding initiation. An alternative method based on analyzing IR difference spectra was also introduced to obtain thermodynamic properties of beta-sheets, which complements the commonly used circular dichroism (CD) and fluorescence techniques. Equilibrium IR measurements indicate that the thermal unfolding of this beta-hairpin is fairly broad. However, it can be described by a two-state transition with a thermal melting temperature of approximately 29 degrees C. Time-resolved IR measurements following a T-jump, probed at 1634 cm(-1), indicate that the folding of this beta-hairpin follows first-order kinetics and is amazingly fast. At 300 K, the folding time is approximately 0.8 micros, which is only 2-3 times slower than that of alpha-helix formation. Additionally, the energetic barrier for folding is small (approximately 2 kcal mol(-1)). These results, in conjunction with results from other studies, support a view that the details of native contacts play a dominant role in the kinetics of beta-hairpin folding.

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (PheCN), could be used as an infrared reporter of local environment. Here, we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using PheCN as a practical fluorescent probe, we studied the binding of calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single PheCN residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the PheCN fluorescence. A series of stochiometric titrations further allowed us to determine the binding affinity (Kd) of this peptide to CaM. Taken together, these results indicated that the PheCN fluorescence is sensitive to environment and could be applicable to a wide variety of biological problems.

The photophysical properties of several 2-substituted, 10-alkylated phenothiazines were measured in several solvents to investigate the relevance of the molecular structure in their photophysics and consequent photochemistry. Because the interaction modes of each drug and its corresponding species strongly depend on the variety of microenvironments in the cells, the properties of each one of these species must also be determined separately to understand fully the mechanism of action of the drug and the mechanism of its side effects. Information on the chemical interactions of the different species at the cellular level can be inferred from the corresponding electronic properties. In this work, we present absorption, steady-state, and time-resolved emission, laser flash photolysis, and quantum theoretical results for the ground state, the first excited singlet and triplet states, and the cation radical of promazine hydrochloride (PZ), 2-chlorpromazine hydrochloride (CPZ), 2-trifluoromethylpromazine hydrochloride (TFMPZ), 2-trifluoromethylperazine dihydrochloride (TFMP), 2-thiomethylpromazine (TMPZ), and thioridazine hydrochloride (TR). The corresponding nonalkylated phenothiazines are included as references. The photophysical properties of this drug family depend more on the solvent and the 2-substituents than on the dialkylaminopropyl chain. The largest effect was found for the triplet state of the 2-halogenated derivatives in phosphate buffer (PBS). Both the quantum yield and the lifetime of this intermediate drop to less than 5% of the corresponding value in organic solvents. The triplet state of halogenated promazines is efficiently quenched by a proton-transfer mechanism, and the rate of this quenching correlates very well with the phototoxicity of the promazine drugs. Therefore, we postulate that this species is directly related to the phototoxic side effect of neuroleptic drugs.

Alpha(1)-antitrypsin (AT) is the most abundantly circulating human proteinase inhibitor in the serpin family. The polymerization of AT, leading to alpha(1)-antitrypsin deficiency, has been studied extensively in vitro by a variety of ensemble methods. Here we report the use of fluorescence correlation spectroscopy to gain further insight into this process. Measurements of the distributions of diffusion times of polymerizing AT, carried out at 45, 50, and 55 degrees C, clearly show the existence of a kinetic lag phase, during which short oligomers are formed, prior to the formation of heterogeneous mixtures of longer polymers, and suggest that long polymers, which appear to be metastable, are produced through the condensation of shorter oligomers.

We identify distinct site-specific dynamics over the time course of Aβ1-23 amyloid formation by using an unnatural amino acid, p-cyanophenylalanine, as a sensitive fluorescent and Raman probe. Our results also suggest the key role of an edge-to-face aromatic interaction in the conformational conversion to form and stabilize β-sheet structure.

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Photophysical and electrochemical properties of protriptyline hydrochloride (PTL-HCl) and its free base were measured in different solvents. Ground-state properties remain unchanged as a function of solvent or protonation state. The fluorescence quantum yield and lifetime, on the other hand, are solvent-dependent. Fluorescence lifetimes results were obtained from monoexponential decay fittings. Characterization of the 266 nm nanosecond laser flash photolysis species reveals the presence of the triplet-triplet transient intermediate at low-intensity conditions. At high laser intensities, two new transient intermediates were observed, which were assigned to the solvated electron and the radical cation. These species are formed through a biphotonic route, as shown with photonicity studies. The relatively high oxidation potentials measured for PTL-HCl and its free base corroborate the biphotonic process required for these species. These electrochemical studies also show that PTL-HCl is easier to oxidize than its free base. Singlet oxygen is established as one of the transient species following triplet-state formation. The PTL-HCl triplet state is probably involved in the dimer formation and other reactive intermediates, and therefore, it might be directly associated with the in vivo phototoxic effects observed for this drug.