Abstract
Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. Previous studies have identified Gly(47) and Gly(51) as possible hinge points in the structure of the protein, allowing backbone segments around the chromophore to undergo large concerted motions. We have designed, crystallized, and solved the structures of three mutants: G47S, G51S, and G47S/G51S. The protein dynamics of these mutants are significantly affected. Transitions in the photocycle, measured with laser induced transient absorption spectroscopy, show rates up to 6-fold different from the wild type protein and show an additive effect in the double mutant. Compared with the native structure, no significant conformational differences were observed in the structures of the mutant proteins. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly(51) may also play a role in PAS domain proteins where it is one of the few conserved residues.
Highlights
Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described
The structures of native photoactive yellow protein (PYP) crystallized in two different space groups (P63 versus P65) were compared, and it was found that the backbones superimposed with an r.m.s.d. of 0.5 Å [21]
Photocycle Kinetics—We have studied the effects of the mutations on the photocycle dynamics of PYP with transient absorption spectroscopy, using an experimental set-up described recently in detail elsewhere [38] that can reach a time-resolution of 10 ns
Summary
Crystallographic and spectroscopic analyses of three hinge-bending mutants of the photoactive yellow protein are described. We conclude that the structural and dynamic integrity of the region around these mutations is of crucial importance to the photocycle and suggest that the hinge-bending properties of Gly may play a role in PAS domain proteins where it is one of the few conserved residues. After absorption of a blue photon, the protein returns from the primary excited state into the first transient groundstate (8 –11), a strongly red-shifted intermediate [12], at the ps time scale. A more moderately red-shifted intermediate (pR), absorbing maximally at 465 nm, is formed on the ns time scale [13]. The pB intermediate subsequently relaxes back to pG on a sub-s time scale [10, 13,14,15] or faster in a light-dependent reaction [16]
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