Peptide GFSKAELAKARAAKRGGY folds in an α-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V, Blanco FJ, Serrano L, 1995, Struct Biol 2:380–385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20° and backbone φ,ψ bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Cα-H, Cα-Cβ, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CβH2 and CγH motions are also correlated and as motionally restricted as backbone CαH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cβ-Cγ bond and phenyl ring fluctuate more simply about the Cα-Cβ bond. These data support the Daragan–Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378–8388) and contribute to a general understanding of internal motions in peptides and proteins.

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine α-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: α-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.