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.

Diffuse reflectance infrared Fourier transform spectroscopic (DRIFTS) measurements (4000-1500 cm(-1)) and the results of neutron powder diffraction have been combined to study the structure of adsorption complexes of water in a NaX zeolite at different water loadings (25, 48, 72, and 120 water molecules per unit cell, respectively). Sharp bands corresponding to non-hydrogen-bonded OH groups of water molecules and broad associate bands due to hydrogen-bonded molecules are observed in the DRIFT spectra. We observe a remarkable downshift of the high-frequency associate band in a narrow temperature interval when the water amount decreases from 120 to 72 molecules per unit cell, which could signify some kind of "phase transition" for the water inside the zeolite cavities. Neutron powder diffraction results show that water molecules are predominantly localized in or near the 12-ring windows. Water molecules with hydrogen-bonded and non-hydrogen-bonded OH groups were found, in agreement with the observation of sharp and broad bands in the DRIFT spectra. We find strong evidence for the formation of cyclic hexamers of water molecules localized in the 12-ring windows, which are further stabilized by hydrogen bonds to framework oxygen atoms.

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