This work reports an explanation for the unusual monoexponential fluorescence decay of 5-fluorotryptophan (5FTrp) in single-Trp mutant proteins [Broos, J.; Maddalena, F.; Hesp, B. H. J. Am. Chem. Soc. 2004, 126, 22-23] and substantially clarifies the origin of the ubiquitous nonexponential fluorescence decay of tryptophan in proteins. Our results strongly suggest that the extent of nonexponential fluorescence decay is governed primarily by the efficiency of electron transfer (ET) quenching by a nearby amide group in the peptide bond. Fluoro substitution increases the ionization potential (IP) of indole, thereby suppressing the ET rate, leading to a longer average lifetime and therefore a more homogeneous decay. We report experimental IPs for a number of substituted indoles including 5-fluoroindole, 5-fluoro-3-methylindole, and 6-fluoroindole, along with accurate ab initio calculations of the IPs for these and 20 related molecules. The results predict the IP of 5-fluorotryptophan to be 0.19 eV higher than that of tryptophan. 5-Fluoro substitution does not measurably alter the excitation-induced change in permanent dipole moment nor does it change the fluorescent state from 1La to 1Lb. In combination with electronic structure information this argues that the increased IP and the decreased excitation energy of the 1La state, together 0.3 eV, are solely responsible for the strong reduction of electron transfer quenching. 6-Fluoro substitution is predicted to increase the IP by a mere 0.09 eV. In agreement with our conclusions, the fluorescence decay curves of 6-fluorotryptophan-containing proteins are well fit using only two decay times compared to three required for Trp.

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When referring to a domain in a multidomain EII we use the terminology`A domain',`B domain' etc. When referring to a domain of EII which has been subcloned and expressed as a separate protein we refer to it as, for example,`IIA mtl ',`IIB glc ', etc.2 Notation : heterodimers formed by mixing or co-expressing two proteins are denoted protein 1/protein 2 (i.e., wild-type/H554A or C384S/H195A). Double mutants are denoted as mutation1:mutation2 (i.e., C384S:H195A).

Papain, modified at Cys-25 with a monodentate phosphite ligand and complexed with Rh(COD)2BF4, is an active catalyst in the hydrogenation of methyl 2-acetamidoacrylate.

Tryptophan, when in a protein, typically shows multiexponential fluorescence decay kinetics. Complex kinetics prevents a straightforward interpretation of time-resolved fluorescence protein data, particularly in anisotropy studies or if the effect of a dynamic quencher or a resonance energy transfer (RET) acceptor is investigated. Here, time-resolved fluorescence data are presented of an isosteric tryptophan analogue, 5-fluorotryptophan, which when biosynthetically incorporated in proteins shows monoexponential decay kinetics. Data are presented indicating that the presence of a fluoro atom at the 5-position suppresses the electron transfer rate from the excited indole moiety to the peptide bond. This process has been related to the multiexponential fluorescence decay of tryptophan in proteins. The monoexponential decay of 5-fluorotryptophan makes it possible to measure simultaneously multiple distances between 5-fluorotryptophan and a RET acceptor. We demonstrate that for an oligomeric protein, consisting of two single-tryptophan-containing subunits, the individual distances between 5-fluorotryptophan and the single substrate binding site can be resolved using a substrate harboring a RET acceptor.

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A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system Dijkstra, Durk; Broos, Jaap; Lolkema, Julius; Enequist, H.; Minke, W.; Robillard, G.T. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document VersionPublisher's PDF, also known as Version of record Publication date : 1996 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Dijkstra, D., Broos, J., Lolkema, J. S., Enequist, H., Minke, W., & Robillard, G. T. (1996). A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvatedependent mannitol transport system. Biochemistry, 35(21), 6628-6634. DOI: 10.1021/bi952222t Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. ReceiVed September 18, 1995; ReVised Manuscript ReceiVed March 14, 1996 X ABSTRACT: The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant . The steady-state fluorescence spectra of EII mtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I -quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I -. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 385 or ...