Fluorenscence depolarization study of internal tryptophan mobility in hydrated azurin films

The effect of hydration on protein dynamics was investigated using steady state fluorescence depolarization on azurin, which has a single tryptophan residue well-buried in the hydrophobic interior. Azurin was imbedded in a thin, solid, water-permeable polymer film. This procedure inhibited whole-protein motion while allowing examination of internal degrees of rotational freedom in the protein as a function of film hydration. In the dry film at room temperature the tryptophan fluorescence emission showed the limiting polarization characteristic of low temperature solutions in which all motion is frozen. As the hydration was increased, the observed polarization value changed sharply at about 0.6 h (h =g water/g film), indicating an increase in the rotational mobility of the tryptophan. The fully hydrated film had a polarization equal to that of an azurin solution measurement extrapolated to infinite viscosity where the rotation of the protein as a whole 1s hindered. The conclusion is that the protein internal mobility is linked to the hydration level, and that hydration can turn on the full protein flexibility on the nanosecond time scale accessible by fluorescence techniques.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physics

Year dc:date

2011

Author dc:creator

• Limkeman, Mark Kenneth

Contributors dc:contributor

• Gratton, E.

• fluorescence depolarization
• internal tryptophan mobility
• hydrated azurin films
• protein dynamics

Statement dc:rights

• 1984 Mark Kenneth Limkeman

Language dc:language

en

Identifier

826568

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/25339