Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

RSFPs (reversibly switchable fluorescent proteins) may be repeatedly converted between a fluorescent and a non-fluorescent state by irradiation and have attracted widespread interest for many new applications. The RSFP Dronpa may be switched with blue light from a fluorescent state into a non-fluorescent state, and back again with UV light. To obtain insight into the underlying molecular mechanism of this switching, we have determined the crystal structure of the fluorescent equilibrium state of Dronpa. Its bicyclic chromophore is formed spontaneously from the Cys62-Tyr63-Gly64 tripeptide. In the fluorescent state, it adopts a slightly non-coplanar cis conformation within the interior of a typical GFP (green fluorescent protein) b-can fold. Dronpa shares some structural features with asFP595, another RSFP whose chromophore has previously been demonstrated to undergo a cis-trans isomerization upon photoswitching. Based on the structural comparison with asFP595, we have generated new Dronpa variants with an up to more than 1000-fold accelerated switching behaviour. The mutations which were introduced at position Val157 or Met159 apparently reduce the steric hindrance for a cis-trans isomerization of the chromophore, thus lowering the energy barrier for the blue light-driven on-to-off transition. The findings reported in the present study support the view that a cis-trans isomerization is one of the key events common to the switching mechanism in RSFPs.

Original publication

DOI

10.1042/BJ20061401

Type

Journal article

Journal

Biochem J

Publication Date

15/02/2007

Volume

402

Pages

35 - 42

Keywords

Amino Acid Sequence, Animals, Crystallization, Crystallography, X-Ray, Fluorescent Dyes, Isomerism, Kinetics, Luminescent Proteins, Models, Molecular, Molecular Sequence Data, Pectinidae, Protein Conformation, Protein Structure, Tertiary, Sea Anemones, Sequence Alignment