Topics in Fluorescence Spectroscopy. Volume 3 Biochemical Applications

Transcription

1 Topics in Fluorescence Spectroscopy Volume 3 Biochemical Applications

2 Topics in Fluorescence Spectroscopy Edited by JOSEPH R. LAKOWICZ Volume 1: Techniques Volume 2: Principles Volume 3: Biochemical Applications

3 Topics in Fluorescence Spectroscopy Volume 3 Biochemical Applications Edited by JOSEPH R. LAKOWICZ Center for Fluorescence Spectroscopy Department of Biological Chemistry University of Maryland School of Medicine Baltimore, Maryland KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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5 Contributors S. Arnold Microparticle Photophysics Laboratory (MP 3 L), Department of Physics, Polytechnic University, Brooklyn, New York Daniel Axelrod Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan A. P. Demchenko A. V. Palladin Institute of Biochemistry of the Academy of Sciences, Kiev , Ukraine L. M. Folan Microprarticle Photophysics Laboratory (MP 3 L), Department of Physics, Polytechnic University, Brooklyn, New York Bryant S. Fujimoto Department of Chemistry, University of Washington, Seattle, Washington Robert M. Fulbright Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan Edward H. Hellen Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan William R. Laws Department of Biochemistry, Mount Sinai School of Medicine, New York, New York Thomas M. Li Development Department, Syva, Palo Alto, California Richard F. Parrish Development Department, Syva, Palo Alto, California J. B. Alexander Ross Department of Biochemistry, Mount Sinai School of Medicine, New York, New York Kenneth W. Rousslang Department of Chemistry, University of Puget Sound, Tacoma, Washington J. Michael Schurr Department of Chemistry, University of Washington, Seattle, Washington v

6 vi Contributors Lu Song Department of Chemistry, University of Washington, Seattle, Washington Christopher D. Stubbs Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania Jane M. Vanderkooi Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Brian Wesley Williams Department of Chemistry, Bucknell University, Lewisburg, Pennsylvania Pengguang Wu Department of Chemistry, University of Washington, Seattle, Washington Herman R. Wyssbrod Department of Chemistry, University of Louisville, Louisville, Kentucky 40292

7 Preface Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. Because of these numerous developments and the rapid appearance of new methods, it has become difficult to remain current on the science of fluorescence and its many applications. Consequently, I have asked the experts in particular areas of fluorescence to summarize their knowledge and the current state of the art. This has resulted in the initial three volumes of Topics in Fluorescence Spectroscopy, which is intended to be an ongoing series which summarizes, in one location, the vast literature on fluorescence spectroscopy. These first three volumes are designed to serve as an advanced text. These volumes describe the more recent techniques and technologies (Volume 1), the principles governing fluorescence and the experimental observables (Volume 2), and applications in biochemistry and biophysics (Volume 3). Additional volumes will be published as warranted by further advances in this field. I welcome your suggestions for future topics or volumes, offers to contribute chapters on specific topics, or comments on the present volumes. Finally, I thank all the authors for their patience with the delays incurred in release of the first three volumes. Baltimore, Maryland Joseph R. Lakowicz vii

8 Contents 1. Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides J. B. Alexander Ross, William R. Laws, Kenneth W. Rousslang, and Herman R. Wyssbrod 1.1. Historical Perspective and Background The Absorption Properties of Tyrosine The Excited Singlet and Triplet States of Tyrosine and Tyrosinate The Zero-Field Splittings of the Triplet State Excited-State Decay Kinetics Quenching Mechanisms of Tyrosine Emission in Polypeptides and Proteins The Peptide Bond Singlet-Singlet and Triplet-Triplet Resonance Energy Transfer Disulfide Bonds and Sulfhydryl Groups Interactions with lonizable Side Chains and Proton Acceptors Emission from Polypeptides and Proteins Fluorescence of Tyrosine Fluorescence of Tyrosinate Phosphorescence and ODMR of Proteins and Polypeptides Tyrosine as an Excited-State Probe for Conformation and Dynamics References Fluorescence and Dynamics in Proteins A. P. Demchenko 2.1. Introduction Dynamics in Proteins Structural Hierarchy and Degrees of Mobility ix

9 x Contents DistributionofMicrostates Analysis of Motions Using Time-Resolved Methods Decay and Quenching of Fluorescence Emission Decay Kinetics Fluorescence Quenching by Intrinsic Quenchers Fluorescence Quenching by Extrinsic Quenchers Rotation of Aromatic Groups Fluorescence Polarization Studies with and without Time Resolution Models of Rotations Fluorescence Spectroscopy of Molecular Relaxation Dynamic Reorientation of Dipoles in the Fluorophore Environment The Two-State Model of Relaxation Continuous Model of Relaxation Site-Photoselection Model Molecular Relaxation and Dynamics of Dipoles in the Protein Globule Relaxational Shift of Steady-State Spectra Time-Resolved Spectra Red-Edge Excitation Spectroscopy Conclusion and Future Prospects References Tryptophan Phosphorescence from Proteins at Room Temperature Jane M. Vanderkooi 3.1. Background Triplet State Formation and Disappearance Energy Diagram General Considerations of Phosphorescence Yield Measurement of Phosphorescence Tryptophan Phosphorescence Emission from Proteins Comparison of Fluorescence and Phosphorescence Emission Spectra Delayed Fluorescence Lifetime of Tryptophan Phosphorescence in Proteins What Affects the Phosphorescence Lifetime? Phosphorescence Quenching by External Molecules Phosphorescence Lifetimes to Measure Conformational Changes in Proteins Phosphorescence Anisotropy and Rotational Motion

10 Contents xi Phosphorescence Anisotropy Anisotropy to Study Proteins Tryptophan Phosphorescence from Cells Conclusions References Fluorescence Studies of Nucleic Acids: Dynamics, Rigidities, and Structures J. Michael Schurr, Bryant S. Fujimoto, Pengguang Wu, and Lu Song 4.1. Introduction Rotational Dynamics of DNA Background Pertinent Questions and Problems Theory Instrumentation Protocol and Data Analysis Experimental Results Rotational Dynamics of DNA in Nucleosomes, Chromatin, Viruses, and Sperm Nucleosomes Chromatin Viruses Sperm Steady-State Studies of DNA Dynamics DNA Dynamics by Fluorescence Microscopy Dynamics of trnas Ethidium Fluorescence Wyebutine Fluorescence Summary and Outlook References Fluorescence in Membranes Christopher D. Stubbs and Brian Wesley Williams 5.1. Introduction Fluorescence Lifetimes The Use of Fluorescence Lifetimes for Membrane Organizational Studies Fluorescence Lifetime Distributions Excimer Probes

11 xii Contents 5.3. Fluorescence Anisotropy Anisotropy Parameters Time-Resolved Anisotropy Applications to Membrane Studies Fluorescent Probes for Lifetime and Anisotropy Studies Fluorescence Energy Transfer Surface Distribution of Fluorophore-Labeled Lipids Location of the Longitudinal and Lateral Position of Membrane Proteins Protein-ProteinAssociations Fluorescence Quenching Determination of Partitioning and Binding of Fluorophore Quenchers to Membranes Location of Fluorophores Solvent Relaxation Surface Charge FutureDirections References Fluorescence and Immunodiagnostic Methods Thomas M. Li and Richard F. Parrish 6.1. Introduction Assay Formats Fluorescence Polarization Immunoassay Substrate-Labeled Fluorescent Immunoassay Intra-Molecularly Quenched Fluorescent Immunoassay Homogeneous Fluorescent Immunoassay in a Dry Reagent Format Fluorescence Excitation Transfer Immunoassay Design of Fluorescent Probes Phycobiliproteins Phase-Resolved Fluorescence Immunoassay Time-Resolved Fluorescence Immunoassay Conclusion References Total Internal Reflection Fluorescence Daniel Axelrod, Edward H. Hellen, and Robert M. Fulbright 7.1. Introduction Theory of TIR Excitation

12 Contents xiii Single Interface Intermediate Layer Emission by Fluorophores near a Surface Description of the Model Mathematical and Physical Basis Graphical Results Theoretical Results for a Distribution of Dipoles: Random Orientations Consequences for Experiments TIRF for a Microscope Inverted Microscope Upright Microscope Prismless TIRF TIRF Interference Fringes General Experimental Suggestions Applications of TIRF Binding of Proteins and Probes to Artificial Surfaces Concentration of Molecules near Surfaces Orientation, Rotation, and Fluorescence Lifetime of Molecules near Surfaces Qualitative Observation of Labeled Cells Fluorescence Energy Transfer and TIRF Reaction Rates at Biosurfaces TIRF Combined with Fluorescence Correlation Spectroscopy (PCS) Summary and Comparisons References Microparticle Fluorescence and Energy Transfer L. M. Folan and S. Arnold 8.1. Introduction Fluorescence from a Microparticle Nature of the Effects Excitation Spectroscopy Interaction of a Plane Wave with a Sphere Excitation of a Dipole and Photoselection Experiments Emission Spectroscopy Interaction between an Excited Electronic State and a Microsphere: Radiative and Nonradiative Decay Rates Angular Intensity Distribution

13 xiv Contents Energy Transfer Experiments Conclusions References Index