No abstract

UV resonance Raman spectroscopy (UVRR) is a powerful method that has the requisite selectivity and sensitivity to incisively monitor biomolecular structure and dynamics in solution. In this perspective, we highlight applications of UVRR for studying peptide and protein structure and the dynamics of protein and peptide folding. UVRR spectral monitors of protein secondary structure, such as the Amide III 3 band and the C α -H band frequencies and intensities can be used to determine Ramachandran Ψ angle distributions for peptide bonds. These incisive, quantitative glimpses into conformation can be combined with kinetic T-jump methodologies to monitor the dynamics of biomolecular conformational transitions. The resulting UVRR structural insight is impressive in that it allows differentiation of, for example, different α-helix-like states that enable differentiating π-and 3 10 -states from pure α-helices. These approaches can be used to determine the Gibbs free energy landscape of individual peptide bonds along the most important protein (un)folding coordinate. Future work will find spectral monitors that probe peptide bond activation barriers that control protein (un)folding mechanisms. In addition, UVRR studies of sidechain vibrations will probe the role of side chains in determining protein secondary, tertiary and quaternary structures. KeywordsProtein folding; melting dynamics; T-jump; UV resonance Raman spectroscopy; UVRR transient spectra; protein (un)folding kinetics The protein folding problemAn understanding of the mechanism(s) of protein folding, whereby the ribosome synthesized biopolymer folds into its native protein is arguably one of the most important unsolved problem in biology. [1][2][3][4][5][6][7] The primary sequence of many or most proteins encodes both the native structure as well as the folding mechanism pathway to the native structure. [8][9][10] An understanding of the encoded protein folding "rules" would dramatically speed insight into protein structure and function.A number of mechanisms have been proposed that differ in the order of folding events. The framework model 11 and the diffusion-collision model 12 propose that the initial step in folding involves formation of native-like secondary structural units, while the hydrophobic collapse model and the nucleation-condensation model 13 suggest that hydrophobic or nucleating domains fold first, and that these structures drive the subsequent formation of secondary structure. Recent energy landscape models 14 propose the occurrence of funnelshaped folding energy landscapes, where the native state is accessed via a strategically * To whom correspondence should be addressed asher@pitt.edu NIH Public Access Thus, tuning the UVRR excitation wavelengths, allows the probing of different chromophoric segments of a macromolecule. Another advantage of deep UV Raman measurements is that there is no interference from molecular relaxed fluorescence. 42 In addition, UVRR can also be used in pump-probe measurements to give kinetic information on ...

In this study, we report for the first time the use of silica-coated superparamagnetic iron oxide nanoparticles (SPION) as contrast agents in biomedical photoacoustic imaging. Using frequency-domain photoacoustic correlation (the photoacoustic radar), we investigated the effects of nanoparticle size, concentration and biological media (e.g. serum, sheep blood) on the photoacoustic response in turbid media. Maximum detection depth and the minimum measurable SPION concentration were determined experimentally. The nanoparticle-induced optical contrast ex vivo in dense muscular tissues (avian pectus and murine quadricept) was evaluated and the strong potential of silica-coated SPION as a possible photoacoustic contrast agents was demonstrated.

Thymine is one of the pyrimidine nucleobases found in DNA. Upon absorption of UV light, thymine forms a number of photoproducts, including the cyclobutyl photodimer, the pyrimidine pyrimidinone [6-4] photoproduct and the photohydrate. Here, we use UV resonance Raman spectroscopy to measure the initial excited-state structural dynamics of the N(1)-substituted thymine derivatives N(1)-methylthymine, thymidine, and thymidine 5'-monophosphate in an effort to understand the role of the N1 substituent in determining the excited-state structural dynamics and the subsequent photochemistry. The UV resonance Raman spectrum of thymidine and thymidine 5'-monophosphate are similar to that of thymine, suggesting that large masses at N(1) effectively isolate the vibrations of the nucleobase. However, the UV resonance Raman spectrum of N(1)-methylthymine is significantly different, suggesting that the methyl group couples into the thymine ring vibrations. The resulting resonance Raman intensities and absorption spectra are self-consistently simulated with a time-dependent expression to quantitatively extract the initial excited-state slopes, homogeneous and inhomogeneous linewidths, and electronic parameters. These results are discussed in the context of the known photochemistry of thymine and its derivatives.

Cytosine is a nucleobase found in both DNA and RNA, while uracil is found only in RNA. Uracil has abstractable protons at N3 and N1. Cytosine has only one abstractable proton at N1 but can also accept a proton at N3. The pKa values of these protons are well-known, but the effect of the change in protonation on the rest of the molecule is not well understood and is very important in base stacking, base pairing, and protein-nucleic acid interactions. In this paper, UV resonance Raman (UVRR) spectroscopy is used to probe the structures of both cytosine and uracil at varying pH to determine the structural changes that take place. The results show that cytosine has increased electronic delocalization when moving to either basic or acidic environments, whereas uracil shows no significant change in acidic environment but increases its electronic delocalization in basic environment.