MUTATIONS IN TRANSHYDROGENASE CHANGE THE FLUORESCENCE EMISSION STATE OF TRP-72 FROM 1La to 1Lb
Karina Tveen Jensen 1, giovanni Strambini 2, Margherita Gonnelli 2, Jaap Broos 3 and J Baz Jackson 4*
1 University of Birmingham
2 CNR Insituto di Biofysico, Pisa
3 University of Groningen
4 Universtiy of Birmingham
* To whom correspondence should be addressed. E-mail: j.b.jackson{at}bham.ac.uk.
Submitted on April 3, 2008
Revised on May 21, 2008
Accepted on 17 June 2008
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Abstract |
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The dI component of Rhodospirillum rubrum transhydrogenase has a single Trp residue (Trp72) which has distinctive optical properties, including short-wavelength fluorescence emission with clear vibrational fine structure, and long-lived, well-resolved phosphorescence emission. We have made a set of mutant dI proteins in which residues contacting Trp72 are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met97 mutants are blue shifted by ~4 nm, giving them a shorter-wavelength emission than any other protein described in the literature, including azurin from Pseudomonas aeruginosa. Fluorescence spectra in low-temperature glasses reveal equivalent well-resolved vibrational bands in wild-type and the mutant dI proteins, and in azurin. Substitution of Met97 in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met97 mutants arises predominantly from the 1Lb excited singlet state of Trp72, whereas 1La is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met97 promotes greater stabilization of 1La than either 1Lb or the ground state. The phosphorescence spectra of Met97 mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the 3La state of the Trp and the ground state.
Key Words:
azurin, fluorescence anisotropy, protein dynamics, transhydrogenase, tryptophan fluorescence, tryptophan phosphorescence
