An unusual red‐edge excitation and time‐dependent Stokes shift in the single tryptophan mutant protein DD‐carboxypeptidase from Streptomyces: The role of dynamics and tryptophan rotamers

The fluorescence emission of the single tryptophan (W233) of the mutant protein DD‐carboxypeptidase from streptomyces is characterized by a red‐edge excitation shift (REES), i.e., the phenomenon that the wavelength of maximum emission depends on the excitation wavelength. This phenomenon is an indication for a strongly reduced dynamic environment of the single tryptophan, which has a very low accessibility to the solvent. The REES shows, however, an unusual temperature and time dependence. This, together with the fluorescence lifetime analysis, showing three resolvable lifetimes, can be explained by the presence of three rotameric states that can be identified using the Dead‐End Elimination method. The three individual lifetimes increase with increasing emission wavelength, indicating the presence of restricted protein dynamics within the rotameric states. This is confirmed by time‐resolved anisotropy measurements that show dynamics within the rotamers but not among the rotamers. The global picture is that of a protein with a single buried tryptophan showing strongly restricted dynamics within three distinct rotameric states with different emission spectra and an anisotropic environment.

[1]  P. K. Mandal,et al.  Excitation-Wavelength-Dependent Fluorescence Behavior of Some Dipolar Molecules in Room-Temperature Ionic Liquids , 2004 .

[2]  Y. Reshetnyak,et al.  Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms. , 2001, Biophysical journal.

[3]  A. Demchenko,et al.  Site selectivity in excited-state reactions in solutions , 1991 .

[4]  Shoshana J. Wodak,et al.  Detection of cavities in a set of interpenetrating spheres , 1991 .

[5]  P. Wahl,et al.  Conformation in the excited state of two tryptophanyl diketopiperazines , 1974 .

[6]  J. Simon.  Time-resolved studies of solvation in polar media , 1988 .

[7]  C. Shank,et al.  Ultrafast Solvation Processes in Polar Liquids Probed with Large Organic Molecules , 1999 .

[8]  A. Demchenko,et al.  Electrochromic modulation of excited-state intramolecular proton transfer: the new principle in design of fluorescence sensors. , 2002, Journal of the American Chemical Society.

[9]  A. Szabo,et al.  Fluorescence decay of tryptophan conformers in aqueous solution , 1980 .

[10]  J M Ghuysen,et al.  2.8-A Structure of penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase from Streptomyces R61 and complexes with beta-lactams. , 1986, The Journal of biological chemistry.

[11]  G. Weber,et al.  Failure of Energy Transfer between Identical Aromatic Molecules on Excitation at the Long Wave Edge of the Absorption Spectrum. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Y. Reshetnyak,et al.  Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues. , 2001, Biophysical journal.

[13]  T. Azumi,et al.  Shift of emission band upon excitation at the long wavelength absorptio edge. 1. A preliminary survey for quinine and related compounds , 1973 .

[14]  W. Galley,et al.  Role of heterogeneity of the solvation site in electronic spectra in solution. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Y. Reshetnyak,et al.  Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins. , 2001, Biophysical journal.

[16]  T. Azumi,et al.  Shift of the emission band upon excitation at the long wavelength absorption edge. II. Importance of the solute–solvent interaction and the solvent reorientation relaxation process , 1975 .

[17]  P. K. Mandal,et al.  Fluorescence studies in a pyrrolidinium ionic liquid: polarity of the medium and solvation dynamics. , 2005, The journal of physical chemistry. B.

[18]  S. Kinoshita,et al.  Dynamics of fluorescence of a dye molecule in solution , 1988 .

[19]  A. Chattopadhyay,et al.  Red Edge Excitation Shift of a Deeply Embedded Membrane Probe: Implications in Water Penetration in the Bilayer , 1999 .

[20]  P. Adams,et al.  Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides. , 2002, Journal of the American Chemical Society.

[21]  Marc De Maeyer,et al.  The Dead-End Elimination Theorem: , 2000 .

[22]  Alexander P Demchenko,et al.  The red-edge effects: 30 years of exploration. , 2002, Luminescence : the journal of biological and chemical luminescence.

[23]  A. P. Demchenko.  [Dependence of human serum albumin fluorescence spectrum on the excitation wavelength]. , 1981, Ukrainskii biokhimicheskii zhurnal.

[24]  G. Weber.  Theory of differential phase fluorometry: Detection of anisotropic molecular rotations , 1977 .

[25]  Lennart Nilsson,et al.  Molecular origin of time-dependent fluorescence shifts in proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[26]  A. Ladokhin,et al.  Red-edge-excitation fluorescence spectroscopy of indole and tryptophan , 2004, European Biophysics Journal.

[27]  A. Chakrabarti,et al.  Organization and dynamics of tryptophan residues in erythroid spectrin: Novel structural features of denatured spectrin revealed by the wavelength‐selective fluorescence approach , 2003, Protein science : a publication of the Protein Society.

[28]  C. Margulis,et al.  Heterogeneity in a room-temperature ionic liquid: persistent local environments and the red-edge effect. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Hellings,et al.  Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain , 2004, Proteins.

[30]  J. M. Morris,et al.  Nonexponential fluorescence decay of aqueous tryptophan and two related peptides by picosecond spectroscopy. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[31]  J. Frère,et al.  Importance of the two tryptophan residues in the Streptomyces R61 exocellular DD-peptidase. , 1992, The Biochemical journal.

[32]  T. Azumi,et al.  Shift of emission band upon the excitation at the long wavelength absorption edge. III. Temperature dependence of the shift and correlation with the time dependent spectral shift , 1976 .

[33]  J. Frère,et al.  Penicillin-sensitive enzymes in peptidoglycan biosynthesis. , 1985, Critical reviews in microbiology.

[34]  Shoshana J. Wodak,et al.  Interactive computer animation of macromolecules , 1984 .

[35]  M. Maroncelli,et al.  Dipole Solvation in Nondipolar Solvents: Experimental Studies of Reorganization Energies and Solvation Dynamics† , 1996 .

[36]  I Lasters,et al.  The dead-end elimination theorem: mathematical aspects, implementation, optimizations, evaluation, and performance. , 2000, Methods in molecular biology.

[37]  Johan Desmet,et al.  The dead-end elimination theorem and its use in protein side-chain positioning , 1992, Nature.

[38]  G. Fleming,et al.  On the origin of nonexponential fluorescence decay in tryptophan and its derivatives , 1983 .

[39]  G. Fleming,et al.  Nonexponential Fluorescence Decay of Tryptophan, Tryptophylglycine, and Glycyltryptophan , 1983 .

[40]  A. Gronenborn,et al.  Nanosecond relaxation dynamics of protein GB1 identified by the time-dependent red shift in the fluorescence of tryptophan and 5-fluorotryptophan. , 2006, The journal of physical chemistry. B.

[41]  Samuel L. C. Moors,et al.  Tryptophan rotamers as evidenced by X-ray, fluorescence lifetimes, and molecular dynamics modeling. , 2006, Biophysical journal.

[42]  M. Hellings,et al.  The dead-end elimination method, tryptophan rotamers, and fluorescence lifetimes. , 2003, Biophysical journal.

[43]  J. Lakowicz,et al.  Red-edge excitation of fluorescence and dynamic properties of proteins and membranes. , 1984, Biochemistry.

[44]  A. Demchenko.  Red-edge-excitation fluorescence spectroscopy of single-tryptophan proteins , 2004, European Biophysics Journal.