Acrylonitrile quenching of trp phosphorescence in proteins: a probe of the internal flexibility of the globular fold.

Quenching of Trp phosphorescence in proteins by diffusion of solutes of various molecular sizes unveils the frequency-amplitude of structural fluctuations. To cover the sizes gap between O(2) and acrylamide, we examined the potential of acrylonitrile to probe conformational flexibility of proteins. The distance dependence of the through-space acrylonitrile quenching rate was determined in a glass at 77 K, with the indole analog 2-(3-indoyl) ethyl phenyl ketone. Intensity and decay kinetics data were fitted to a rate, k(r) =k(0) exp[-(r -r(0))/r(e)], with an attenuation length r(e) = 0.03 nm and a contact rate k(0) = 3.6 x 10(10) s(-1). At ambient temperature, the bimolecular quenching rate constant (kq) was determined for a series of proteins, appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to kq = 1.9 x 10(9) M(-1)s(-1) for free Trp in water, in proteins kq ranged from 6.5 x 10(6) M(-1)s(-1) for superficial sites to 1.3 x 10(2) M(-1)s(-1) for deep cores. The short-range nature of the interaction and the direct correlation between kq and structural flexibility attest that in the microsecond-second timescale of phosphorescence acrylonitrile readily penetrates even compact protein cores and exhibits significant sensitivity to variations in dynamical structure of the globular fold.