INTRODUCTION OF A TRP RESIDUE INTO ALPHA-4 AS A PROBE OF DYNAMICS

To investigate the dynamic properties of alpha4, a designed four-helix bundle protein, a Leu residue sequestered in the hydrophobic core of the bundle was changed to Trp. If the interior of the protein was very loosely packed, this replacement should be accommodated quite readily. On the other hand, if the protein was reasonably well-packed (as in the native state or an intermediate late on the folding pathway), the substitution should perturb the packing and destabilize the protein. In fact, the mutation of Leu to Trp was strongly destabilizing, decreasing the free energy of folding by approximately 3-4 kcal/mol. Time-resolved fluorescence measurements suggest that the Trp side chain might be held in two or more distinct orientations in the interior of the protein although it is not held in so many orientations as to average the near-UV signal in the CD spectrum. Finally, fluorescence anisotropy decay showed that the Trp indole side chain was immobilized on the nanosecond time scale, indicating there is a larger barrier for rotation of side chains in the interior of alpha4 as compared to Trp-containing peptides in more fluid media such as homogeneous liquids, SDS micelles, or bilayers.