| Water plays an essential role in the structural dynamics of many proteins. This is apparent from the fact that these dynamics strongly change when the proteins are dehydrated. Recently, evidence was found that the water interacting with proteins possesses special properties: its dynamical and structural properties appear to be strongly different from bulk liquid water\cite{pal02,pal03}. Hence, the water near and inside proteins is often denoted as 'biological' water. A recently developed technique to study the microscopic structure and dynamics of water is femtosecond mid-infrared spectroscopy. This technique has been applied to study the structure and dynamics of bulk liquid water, and of water solvating simple ions \cite{kropman01,omta03}. In this technique the orientational and translational motions of the water molecules are measured by following the frequency and orientation of the OH-stretch vibrations of the water molecules in time with mid-infrared laser pulses. Here we propose to use femtosecond mid-infrared spectroscopy to study the orientational and translational motions of 'biological' water molecules interacting with the prototype proteins Photo-active Yellow Protein (PYP) and Proteorhodopsin \ cite{kandori00a,bergo04}. Both proteins have an important function as a photoreceptor in biological signal transfer. In this project the expertise on femtosecond mid-infrared spectroscopy of aqueous systems of the first applicant will be combined with the expertise on PYP and Proteorhodopsin of the second applicant. Using two-pulse femtosecond mid-IR spectroscopy, we will determine to what extent the dynamics of water solvating PYP and Proteorhodopsin differ from the dynamics of bulk liquid water. In these experiments, the photo-cycle of the proteins will not be triggered, meaning that the equilibrium dynamics of the hydration layers are probed. By studying the water dynamics for proteins at different degrees of hydration, we will determine the range over which the dynamics of the hydration layer differ from bulk water. In addition, we propose to investigate the dynamics of the hydration layers of PYP and Proteorhodopsin after triggering of the functional turn-over with a femtosecond visible pulse. The translational and orientational dynamics of the hydration shell will be measured at different stages of the functional activity using delayed femtosecond mid-infrared pump and probe pulses. These three-pulse (visible-mid-IR-mid-IR)experiments will reveal whether the conformational changes of the protein (that are necessary for functional turnover) are accompanied by changes in the translational and orientational dynamics of the 'biological' water molecules. |
