We investigated the assignments and the conformational dependencies of the UV resonance Raman bands of the 21-residue mainly alanine peptide (AP) and its isotopically substituted derivatives in both their R-helical and PPII states. We also examined smaller peptides to correlate conformation, hydrogen bonding, and structure. Our vibrational mode analysis confirms the complex nature of the amide III region, which contains many vibrational modes. We assign these bands by interpreting the isotopically induced frequency shifts and the conformational sensitivity of these bands and their temperature dependence. Our assignments of the amide bands in some cases agree, but in other cases challenge previous assignments

We report the ultrasensitive detection of adenine using deep-UV surface-enhanced resonance Raman scattering on aluminum nanostructures. Well-defined Al nanoparticle arrays fabricated over large areas using extreme-UV interference lithography exhibited sharp and tunable plasmon resonances in the UV and deep-UV wavelength ranges. Theoretical modeling based on the finite-difference time-domain method was used to understand the near-field and far-field optical properties of the nanoparticle arrays. Raman measurements were performed on adenine molecules coated uniformly on the Al nanoparticle arrays at a laser excitation wavelength of 257.2 nm. With this technique, less than 10 amol of label-free adenine molecules could be detected reproducibly in real time. Zeptomole (~30,000 molecules) detection sensitivity was readily achieved proving that deep-UV surface-enhanced resonance Raman scattering is an extremely sensitive tool for the detection of biomolecules.

Poly (N-isopropylacrylamide) (PNIPAM) is the premier example of a macromolecule that undergoes a hydrophobic collapse when heated above its lower critical solution temperature (LCST). Here we utilize, dynamic light scattering, H-NMR, steady-state and time-resolved UVRR measurements to determine the molecular mechanism of PNIPAM's hydrophobic collapse. Our steady-state results indicate that in the collapsed state the amide bonds of PNIPAM do not engage in inter-amide hydrogen bonding, but are hydrogen bonded to water molecules. At low temperatures, the amide bonds of PNIPAM are predominantly fully water hydrogen bonded, whereas, in the collapsed state one of the two normal C=O hydrogen bonds is lost. The NH-water hydrogen bonding, however, remains unperturbed by the PNIPAM collapse. Our kinetic results indicate a mono-exponential collapse with τ~360 (±85) ns. The collapse rate indicates a persistence length of n~10. At lengths shorter than the persistence length the polymer acts as an elastic rod, whereas, at lengths longer than the persistence length the polymer backbone conformation forms a random coil. Based on these results we propose that at low temperatures PNIPAM adopts an extended, water-exposed conformation that is stabilized by favorable NIPAM-water solvation shell interactions which stabilize large clusters of water molecules. At elevated temperatures, thermal agitation disrupts these interactions. The PNIPAM+water polymer undergoes a volume phase transition, expels water and shrinks to a compact conformation that reduces its hydrophobic solvent accessible surface area. In this compact state, PNIPAM forms small hydrophobic nano-pockets where the (i, i +3) isopropyl groups make hydrophobic contacts. A persistent length of n~10 suggests a cooperative collapse where hydrophobic interactions between adjacent hydrophobic pockets stabilize the collapsed PNIPAM.

Trp-cage, a synthetic 20 residue polypeptide, is proposed to be an ultrafast folding synthetic miniprotein which utilizes tertiary contacts to define its native conformation. We utilized UV resonance Raman spectroscopy (UVRS) with 204 and 229 nm excitation to follow its thermal melting. Our results indicate that Trp-cage melting is complex, and it is not a simple two-state process. Using 204 nm excitation we probe the peptide secondary structure and find the Trp-cage's alpha-helix shows a broad melting curve where on average four alpha-helical amide bonds melt upon a temperature increase from 4 to 70 degrees C. Using 229 nm excitation we probe the environment of the Trp side chain and find that its immediate environment becomes more compact as the temperature is increased from 4 to 20 degrees C; however, further temperature increases lead to exposure of the Trp to water. The chi(2) angle of the Trp side chain remains invariant throughout the entire temperature range. Previous kinetic results indicated a single-exponential decay in the 4-70 degrees C temperature range, suggesting that Trp-cage behaves as a two-state folder. However, this miniprotein does not show clear two-state behavior in our steady-state studies. Rather it shows a continuous distribution of steady-state spectral parameters. Only the alpha-helix melting curve even hints of a cooperative transition. Possibly, the previous kinetic results monitor only a small region of the Trp-cage which locally appears two-state. This would then argue for spatially decoupled folding even for this small peptide.

The effect of hydrogen bonding on the amide group vibrational spectra has traditionally been rationalized by invoking a resonance model where hydrogen bonding impacts the amide functional group by stabilizing its [-O-C=NH+] structure over the [O=C-NH] structure. However, Triggs and Valentini’s UV-Raman study of solvation and hydrogen bonding effects on ε-caprolactum, N,N-dimethylacetamide (DMA) and N-methylacetamide (NMA) (J .Phys. Chem., 1992; 96, 6922-31) cast doubt on the validity of this model by demonstrating that contrary to the resonance model prediction, carbonyl hydrogen bonding does not impact the AmII’ frequency of DMA. In this study we utilize density functional theory (DFT) calculations to examine the impact of hydrogen bonding on the C=O and N-H functional groups of NMA, which is typically used as a simple model of the peptide bond. Our calculations indicate that, as expected, the hydrogen bonding frequency dependence of the AmI vibration predominantly derives from the C=O group, whereas the hydrogen bonding frequency dependence of the AmII vibration primarily derives from N-H hydrogen bonding. In contrast, the hydrogen bonding dependence of the conformation sensitive AmIII band derives equally from both C=O and N-H groups, and, thus, is equally responsive to hydrogen bonding at the C=O or N-H site. Our work shows that a clear understanding of the normal mode composition of the amide vibrations is crucial for an accurate interpretation of the hydrogen bonding dependence of amide vibrational frequencies.