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Papers by Kathrin Winkler

Zeitschrift für Physikalische Chemie, 2000

Ultrafast pump-probe experiments with a time-resolution of 30 fs have been carried out to explore... more Ultrafast pump-probe experiments with a time-resolution of 30 fs have been carried out to explore the non-radiative relaxation dynamics of electronically excited 1,8-dihydroxyanthraquinone (DHAQ) in polar liquid solution. The results are discussed in terms of a Lippincott-Schroeder double-minimum potential along the proton-transfer reaction coordinate for the ground (S

Kapitel 1 Einleitung Grün Fluoreszierende Proteine (GFP) können in einer Vielzahl biolumineszente... more Kapitel 1 Einleitung Grün Fluoreszierende Proteine (GFP) können in einer Vielzahl biolumineszenter Coelenterata (Hohltiere) innerhalb des Stammes der Nesseltiere (Hydrozoa/Anthrazoa) gefunden werden 2. Sie wurden erstmals von Shimomura und Mitarbeitern bei der Untersuchung der Biolumineszenz der Qualle Aequorea aequorea entdeckt 3, 4. Die schirmartig erscheinende Qualle besitzt an ihrem Rand Organe, die aus mehr als 120 kleinen Körnchen bestehen und das charakteristische grüne Licht emittieren (Abbildung 1.1). Eine hypsochrome Verschiebung der Emission nach Extraktion des Proteins wies darauf hin, dass für das Auftreten einer grünen Fluoreszenz mehr als nur ein Protein verantwortlich sein muss. Aus der Qualle Aequorea aequorea konnten schließlich das Protein Aequorin und ein weiteres, das Grün Fluoreszierende Protein (GFP) extrahiert werden. Beide Proteine können unter natürlichen Bedingungen nur als gemeinsamer Komplex die für das GFP charakteristische grüne Fluoreszenz erzeugen. 1 Alle natürlich vorkommenden Grün Fluoreszierenden Proteine emittieren Licht in einem Wellenlängenbereich von 490-520 nm. Die meisten weisen ein Emissionsmaximum bei 508-509 nm auf. Die Absorptionsmaxima der jeweiligen Spezies variieren jedoch stärker (395-498 nm) 2. Das einzige im ultravioletten Spektralbereich (λ max = 398 nm) absorbierende, natürlich vorkommende GFP ist das der Qualle Aequorea Victoria 7. Dieses kann durch Klonierung und Expression in größeren Mengen hergestellt werden 8, 9 und ist daher das Grün Fluoreszierende Protein, welches bisher am detailliertesten untersucht wurde.

Ultrafast Phenomena, 2000

Springer Series in Chemical Physics, 2003

The optical response of wild-type green-fluorescent protein (GFP) has been measured at room tempe... more The optical response of wild-type green-fluorescent protein (GFP) has been measured at room temperature following 40-fs, 400-nm photoexcitation. In the spectral range of the stationary fluorescence of GFP, delayed emission due to excited-state proton transfer (ESPT) can be induced. The entire spectro-temporal response is simulated quantitatively using a novel dynamical model which includes an energy-dependent rate constant for ESPT, intra- and intermolecular flow of vibrational energy, and an additional non-radiative decay pathway leading to internal conversion.

GBM Annual Fall meeting Berlin/Potsdam 2005, 2005

Physical Chemistry Chemical Physics, 2002

ABSTRACT

Phys. Chem. Chem. Phys., 2006

The ultrafast internal conversion (IC) dynamics of the carbonyl carotenoid 12 0-apo-b-caroten-12 ... more The ultrafast internal conversion (IC) dynamics of the carbonyl carotenoid 12 0-apo-b-caroten-12 0-al has been investigated in solvents of varying polarity using time-resolved femtosecond transient absorption spectroscopy. The molecules were excited to the S 2 state by a pump beam of either 390 or 470 nm. The subsequent intramolecular dynamics were detected at several probe wavelengths covering the S 0-S 2 and S 1-S n bands. For the S 1-S 0 internal conversion process, a remarkably strong acceleration with increasing polarity was found, e.g., lifetimes of t 1 = 220 ps (n-hexane), 91 ps (tetrahydrofuran) and 8.0 ps (methanol) after excitation at 390 nm. The observation can be rationalized by the formation of a combined S 1 /ICT (intramolecular charge transfer) state in the more polar solvents. The effect is even stronger than the strongest one reported so far in the literature for peridinin. Addition of lithium salts to a solution of 12 0-apob-caroten-12 0-al in ethanol leads only to small changes of the IC time constant t 1. In addition, we estimate an upper limit for the time constant t 2 of the S 2-S 1 internal conversion process of 300 fs in all solvents.

Photochemistry and Photobiology, 2009

The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐h... more The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100, 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light‐activated bR and rhodopsin detected by time‐resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real‐time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin‐II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time‐resolved fluorescence ...

Photochemistry and Photobiology, 2007

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-re... more Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico-to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is lightactivated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPCmodule. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.

Optics Letters, 2005

We report enhancement of two-photon fluorescence (TPF) excitation in fluorescent dyes and fluores... more We report enhancement of two-photon fluorescence (TPF) excitation in fluorescent dyes and fluorescently labeled biomolecules by exploiting the optical properties of double grating waveguide structures (DGWSs). Picosecond laser pulses generate a large evanescent field based on the guided mode phenomenon in the resonant DGWSs, which induces strong TPF signals from fluorescent dyes at the waveguide surface. By recording enhanced TPF signals of Rhodamine B and Lucifer Yellow under resonance conditions, a detection sensitivity of concentrations of approximately one dye molecule per 0.1 m 2 was achieved. For the first time to our knowledge, enhanced TPF signals of a Lucifer Yellow-labeled biomolecule (human self-peptide) in an aqueous environment are demonstrated. These results strongly encourage the use of DGWSs as enhancement platforms in modern biophysics and biotechnology for investigations of biological membranes and cells.

Journal of Biological Chemistry, 2008

A single amino acid exchange between the major histocompatibility complex molecules HLA-B*2705 an... more A single amino acid exchange between the major histocompatibility complex molecules HLA-B*2705 and HLA-B*2709 (Asp-116/His) is responsible for the emergence of distinct HLA-B27restricted T cell repertoires in individuals harboring either of these two subtypes and could correlate with their differential association with the autoimmune disease ankylosing spondylitis. By using fluorescence depolarization and pK a calculations, we investigated to what extent electrostatic interactions contribute to shape antigenic differences between these HLA molecules complexed with viral, self, and non-natural peptide ligands. In addition to the established main anchor of peptides binding to HLA-B27, arginine at position 2 (pArg-2), and the secondary anchors at the peptide termini, at least two further determinants contribute to stable peptide accommodation. 1) The interaction of Asp-116 with arginine at peptide position 5, as found in pLMP2 (RRRWRRLTV; viral) and pVIPR (RRKWRRWHL; self), and with lysine in p⍀, as found in gag (KRWIILGLNK; viral), additionally stabilizes the B*2705 complexes by ϳ5 and ϳ27 kJ/mol, respectively, in comparison with B*2709. 2) The protonation state of the key residues Glu-45 and Glu-63 in the B-pocket, which accommodates pArg-2, affects peptide binding strength in a peptide-and subtype-dependent manner. In B*2705/pLMP2, protonation of Glu-45/Glu-63 reduces the interaction energy of pArg-2 by ϳ24 kJ/mol as compared with B*2705/pVIPR. B*2705/pVIPR is stabilized by a deprotonated Glu-45/Glu-63 pair, evoked by allosteric interactions with pHis-8. The mutual electrostatic interactions of peptide and HLA molecule, including peptide-and subtype-dependent protonation of key residues, modulate complex stability and antigenic features of the respective HLA-B27 subtype.

Biophysical Journal, 2007

Analysis of antigen dissociation provides insight into peptide presentation modes of folded human... more Analysis of antigen dissociation provides insight into peptide presentation modes of folded human leukocyte antigen (HLA) molecules, which consist of a heavy chain, b 2-microglobulin (b 2 m), and an antigenic peptide. Here we have monitored peptide-HLA interactions and peptide dissociation kinetics of two HLA-B27 subtypes by fluorescence depolarization techniques. A single natural amino-acid substitution distinguishes the HLA-B*2705 subtype that is associated with the autoimmune disease ankylosing spondylitis from the non-disease-associated HLA-B*2709 subtype. Peptides with C-terminal Arg or Lys represent 27% of the natural B*2705 ligands. Our results show that dissociation of a model peptide with a C-terminal Lys (GRFAAAIAK) follows a two-step mechanism. Final peptide release occurs in the second step for both HLA-B27 subtypes. However, thermodynamics and kinetics of peptide-HLA interactions reveal different molecular mechanisms underlying the first step, as indicated by different activation energies of 95 6 8 kJ/mol (HLA-B*2705) and 150 6 10 kJ/mol (HLA-B*2709). In HLA-B*2709, partial peptide dissociation probably precedes fast final peptide release, while in HLA-B*2705 an allosteric mechanism based on long-range interactions between b 2 m and the peptide binding groove controls the first step. The resulting peptide presentation mode lasts for days at physiological temperature, and determines the peptide-HLA-B*2705 conformation, which is recognized by cellular ligands such as T-cell receptors.

The Journal of Chemical Physics, 2000

The ultrafast optical Kerr-response of water and heavy water has been measured at 1 bar in the te... more The ultrafast optical Kerr-response of water and heavy water has been measured at 1 bar in the temperature range between 273 and 373 K. The nuclear Kerr response of the liquid exhibits a pronounced double exponential decay on longer time scales after dephasing of impulsively perturbed acoustic modes is completed. The time constant, τ2, characterizing the slowly decaying exponential component of the Kerr-response function is in quantitative agreement with rotational diffusion time constants of the water molecules obtained form nuclear magnetic resonance (NMR) spin-lattice relaxation rates. A detailed comparison with THz time domain spectroscopy demonstrates that the reorientational dynamics responsible for the long time tail of the Kerr response are due to single molecule as opposed to collective effects. Furthermore, a good agreement between the single molecule rotational diffusion and the Stokes–Einstein–Debye equation is found in the temperature range of thermodynamic stability of...