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Tryptophan phosphorescence spectroscopy is one of the most sensitive methodologies around to study the local protein structure. For proteins in buffer, the phosphorescence lifetime (τp) directly reports about the local flexibility of the site with τp ranging from (sub) milliseconds for Trp at a solvent exposed position to several seconds if the Trp side chain is embedded in a rigid protein core. Because of the long intrinsic τp of Trp, 6.5 s, the method is extremely sensitive for quenchers e.g. oxygen and its concentration needs to be reduced to a level of several nM. This extreme sensitivity for quenchers has prevented widespread use of Trp phosphorescence in molecular biology. Substitution of the indole ring with a heavy atom is expected to significantly reduce the intrinsic τp, and thus the sensitivity for quenchers. We measured intrinsic τp values of 400 ms for 6-chloroindole, 13 ms for 6- bromoindole and 38 ms for 5-bromoindole, demonstrating the potential of these Trp analogs as phosphorescence probes in proteins. A straightforward approach to produce proteins labeled with these analogs was not available. We developed before a Trp auxotrophic strain of Lactococcis lactus able to incorporate Trp analogs in proteins which E. coli Trp auxotrophs do not translate. However, this L. lactus strain was found not able to translate bromo- or cloro-substituted Trp analogs. To overcome this, the aminoacyl t-RNA Trp synthetase enzyme from L. lactus was cloned and overexpressed in L. lactus together with a single-Trp containing target protein. This approach resulted in the efficient incorporating (87-91%) of the halogenated Trp analogs and a high protein yield (25-50 mg/L culture). Details of the methodology are presented as well as the spectroscopic characterization of the chloro- and bromo-Trp containing proteins.

The dI component of Rhodospirillum rubrum transhydrogenase has a single Trp residue (Trp(72)), 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 Trp(72) are conservatively substituted. The room-temperature fluorescence-emission spectra of our three Met(97) mutants are blue shifted by approximately 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 show equivalent well-resolved vibrational bands in wild-type and the mutant dI proteins, and in azurin. Substitution of Met(97) in dI changes the relative intensities of some of these vibrational bands. The analysis supports the view that fluorescence from the Met(97) mutants arises predominantly from the (1)L(b) excited singlet state of Trp(72), whereas (1)L(a) is the predominant emitting state in wild-type dI. It is suggested that the sulfur atom of Met(97) promotes greater stabilization of (1)L(a) than either (1)L(b) or the ground state. The phosphorescence spectra of Met(97) mutants are also blue-shifted, indicating that the sulfur atom decreases the transition energy between the (3)L(a) state of the Trp and the ground state.