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The solvent-dependent ground-state conformational equilibrium and excited-state dynamics of 2-(2‘-hydroxyphenyl)benzoxazole have been characterized in several solvents on the femtosecond to nanosecond time scales. The only observable ground-state tautomer is the enol, which exists in equilibrium between the syn- and anti-rotational isomers. In the anti-enol isomer, the phenyl hydroxyl group appears to not interact strongly with solvent but rather forms a strong intramolecular hydrogen bond with the benzoxazole oxygen atom. In the syn-enol isomer, the phenyl hydroxyl proton may interact with solvent or form an internal hydrogen bond with the benzoxazole nitrogen atom. Upon excitation, the proton is transferred from the oxygen atom to the nitrogen atom of the internally hydrogen bonded syn-enol isomer in 170 fs, regardless of the solvent. The lifetime of the resulting excited keto tautomer is solvent dependent and on the order of picoseconds. In addition to these dynamics, several additional dynamic process...

The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl-labeled primer/template indicate that the Q419A, Y423A, and R455A mutations disrupted the ability of the Klenow fragment to melt duplex DNA and bind the frayed terminus at the exonuclease site. In contrast, the N420A mutation stabilized binding of a duplex terminus to the exonuclease site, suggesting that the N420 side chain facilitates the 3'-5' exonuclease reaction by introducing strain into the bound DNA substrate. Together, these results demonstrate that protein side chains that interact with the second or third nucleotides from the terminus can participate in both the chemical step of the exonuclease reaction, by anchoring the substrate in the active site or by ensuring proper geometry of the scissile phosphate, and in the prechemical steps of double-stranded DNA hydrolysis, by facilitating duplex melting.

The Klenow fragment of Escherichia coli DNA polymerase I catalyzes template-directed synthesis of DNA and uses a separate 3'-5' exonuclease activity to edit misincorporated bases. The polymerase and exonuclease activities are contained in separate structural domains. In this study, nine Klenow fragment derivatives containing mutations within the polymerase domain were examined for their interaction with model primer-template duplexes. The partitioning of the DNA primer terminus between the polymerase and 3'-5' exonuclease active sites of the mutant proteins was assessed by time-resolved fluorescence anisotropy, utilizing a dansyl fluorophore attached to the DNA. Mutation of N845 or R668 disrupted favorable interactions between the Klenow fragment and a duplex containing a matched terminal base pair but had little effect when the terminus was mismatched. Thus, N845 and R668 are required for recognition of correct terminal base pairs in the DNA substrate. Mutation of N675, R835, R836, or R841 resulted in tighter polymerase site binding of DNA, suggesting that the side chains of these residues induce strain in the DNA and/or protein backbone. A double mutant (N675A/R841A) showed an even greater polymerase site partitioning than was displayed by either single mutation, indicating that such strain is additive. In both groups of mutant proteins, the ability to discriminate between duplexes containing matched or mismatched base pairs was impaired. In contrast, mutation of K758 or Q849 had no effect on partitioning relative to wild type, regardless of DNA mismatch character. These results demonstrate that DNA mismatch recognition is dependent on specific amino acid residues within the polymerase domain and is not governed solely by thermodynamic differences between correct and mismatched base pairs. Moreover, this study suggests a mechanism whereby the Klenow fragment is able to recognize polymerase errors following a misincorporation event, leading to their eventual removal by the 3'-5' exonuclease activity.

Structural features of the single and mixed micellar systems of sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB) were characterized using the fluorescence probe 6-propionyl-2-(dimethylamino)naphthalene (Prodan). In particular, our investigations capitalized on the spectral sensitivity of Prodan to its environment as well as the extensive solubility of Prodan in solvents of varying polarity and/or hydrophobicity to effectively use a three-mode factor analysis technique to resolve the coincident emission from Prodan in multiple microenvironments of single and mixed micelle systems. Our investigations reveal parameters of Prodan fluorescence that reflect the relative polarities of the surfaces of SDS and DTAB micellar cores, the permeability of the SDS micelle interface, and the heterogeneity of SDS−DTAB mixed micellar systems. In particular, we observe a strong affinity of Prodan for both SDS and DTAB micelles at the water−surfactant interface with the emission λmax of Prodan consist...