Topics in Fluorescence Spectroscopy

by Lakowicz, Joseph R.

• ISBN13: 9780306464515
• ISBN10: 0306464519
• Format: Hardcover
• Copyright: 2001-01-01
• Publisher: Plenum Pub Corp

Summary

This sixth volume in the highly regarded series is an essential addition to libraries serving analytical chemists, spectroscopists, biochemists, and biophysicists. Maintaining the high standards set by its predecessors, Protein Fluorescence presents twelve state-of-the-art chapters by some of the most respected researchers in the field.

Author Biography

Dr. J.R. Lakowicz is Professor of Biochemistry at the University of Maryland School of Medicine and Director of the Center for Fluorescence Spectroscopy. Dr. Lakowicz has published over 400 scientific articles, has edited numerous books, holds 16 issued patents, and is the sole author of the widely used text, Principles of Fluorescence Spectroscopy, also published by Kluwer Academic/Plenum Publishers, now in its Second Edition.

Table of Contents

Intrinsic Fluorescence of Proteins
Maurice R. Eftink
Introduction	1	(1)
Overview	2	(2)
Patterns in Protein Fluorescence	4	(5)
Some Recent Topics	9	(3)
Open Questions	12	(1)
Summary	13	(4)
References	13	(4)
Spectral Enhancement of Proteins by in vivo Incorporation of Tryptophan Analogues
J.B. Alexander Ross
Elena Rusinova
Linda A. Luck
Kenneth W. Rousslang
Introduction	17	(4)
Brief History	19	(2)
In vivo Analogue Incorporation	21	(8)
A General Approach for in vivo Incorporation of Analogues	23	(3)
Analyzing the Efficiency of Analogue Incorporation	26	(3)
Spectral Features of TRP Analogues	29	(8)
Absorption of Analogues	30	(1)
Fluorescence-Analogue Models	31	(2)
Phosphorescence-Analogue Containing Proteins	33	(1)
Phosphorescence - Analogue Models	34	(2)
Phosphorescence - Analogue Containing Proteins	36	(1)
Prospects	37	(6)
References	39	(4)
Room Temperature Tryptophan Phosphorescence as a Probe of Structural and Dynamic Properties of Proteins
Vinod Subramaniam
Duncan G. Steel
Ari Gafni
Introduction	43	(2)
Factors Influencing Tryptophan Phosphorescence in Fluid Solution and in Proteins	45	(3)
Protein Dynamics and Folding Studied Using RTP	48	(5)
Alkaline Phosphatase	48	(3)
Azurin	51	(1)
Beta-Iactoglobulin	51	(1)
Ribonuclease T1	52	(1)
New Developments in RTP for Protein Studies	53	(14)
Distance Measurements using RTP (Diffusion enhanced energy transfer, electron transfer and exchange interactions	53	(2)
H-D Exchange Studies	55	(1)
Circularly Polarized Phosphorescence (CPP)	55	(3)
Stopped Flow RTP	58	(1)
RTP from trp Analogues	58	(1)
Concluding Remarks and Prospects for the future	59	(1)
References	60	(7)
Azurins and Their Site-Directed Mutants
Giampiero Mei
Nicola Rosato
Alessandro Finazzi Agro
A Brief Overview on Azurin and its Dynamic Fluorescence Properties	67	(3)
Experimental Procedures	70	(1)
Copper-Containing Azurins	71	(4)
The Apo-Proteins	75	(4)
Conclusions	79	(4)
References	79	(4)
Barnase: Fluorescence Analysis of a Three Tryptophan Protein
Yves Engelborghs
Alan Fersht
Introduction	83	(2)
Results Obtained by the Method of Subtraction	85	(8)
pH-Dependency of the fluorescence	85	(1)
The Effect of Removing W35	85	(1)
The Effect of Removing W71	86	(1)
The Effect of Removing W94	86	(1)
Calculation of the Absorption and Fluorescence Emission Spectra of the Individual Tryptophans	87	(1)
Calculations of the Forster Energy - Transfer on the Basis of Spectral Data	88	(1)
The Fluorescence Lifetimes	89	(1)
Measured and Calculated Lifetimes	89	(2)
Energy Transfer Calculations Using Lifetime Data	91	(1)
Discussion of Data Obtained from Single Tryptophan Mutants	92	(1)
Characterization of the Double Mutant Protein	93	(3)
Steady-State Fluorescence Parameters	93	(1)
Fluorescence Lifetimes	94	(1)
Calculation of the Fluorescence Decay Parameters of Multi-Tryptophan Proteins from the Emission of Single-Tryptophan Proteins	95	(1)
Fluorescence Anisotropy	96	(1)
Steady-State Phosphorescence	97	(1)
Concentration Dependence of Phosphorescence Intensity	97	(2)
Conclusions	99	(4)
References	100	(3)
Fluorescence Study of the DsbA Protein from Escherichia Coil
Alain Sillen
Jens Hennecke
Rudi Glockshuber
Yves Engelborghs
Introduction	103	(3)
Fluorescence Properties of W76	106	(6)
Fluorescence Properties of W126	112	(3)
Quenching Analysis	112	(2)
Molecular Mechanics	114	(1)
Linking the Conformations with the Lifetimes	114	(1)
Overall Scheme of the Quenching in DBSA	115	(1)
Conclusion	115	(8)
References	119	(4)
The Conformational Flexibility of Domain III of Annexin V is Modulated by Calcium, pH and Binding to Membrane/Water Interfaces
Jaques Gallay
Jana Sopkova
Michael Vincent
Introduction	123	(2)
Experimental Procedures	125	(7)
Protein Preparation and Chemicals	125	(1)
Preparation of Phospholipidic Vescicles and Reverse Micelles	125	(1)
Steady-State Fluorescence Measurements	126	(1)
Time-Resolved Fluorescence Measurements	126	(1)
Analysis of the Time-Resolved Fluorescence Data	127	(1)
Fluorescence Polarized Fluorescence Intensity Decays	127	(1)
Excited State Lifetime Distribution	128	(1)
Rotational Correlation Time Distribution	129	(1)
Wobbling-in-Cone Angle Calculation	130	(1)
Absorbance and Circular Dichroism Measurements	131	(1)
Results	132	(26)
Effect of Calcium on the Structure and Dynamics of Domain III of Annexin V	132	(1)
UV-Difference Absorption Spectra	132	(1)
Circular Dichroism	132	(3)
Steady-State Fluorescence of Trp187	135	(2)
Time-Resolved Fluorescence Intensity Decay of Trp187	137	(2)
Fluorescence Anisotropy of Trp187	139	(4)
Effect of pH on the Conformation and Dynamics of Domain III of Annexin V	143	(1)
Steady-State Fluorescence Emission Spectrum of Trp187	143	(1)
Excited State Lifetime Heterogeneity of Trp187 at Different pH	144	(1)
Time-Resolved Fluorescence Anisotropy Study as a Function of pH	145	(1)
Accessibility of Trp187 to Acrylamide, a Water Soluble Fluorescence Quencher	146	(1)
Secondary Structure of Annexin V as a Function of pH: Circular Dichroism Study	147	(2)
The Interaction of Annexin V with Small Unilamellar Vesicles	149	(1)
Polarity Change Around Trp187 Induced by the Interaction with Membranes: Steady-State Fluorescence Spectra of Trp187	149	(1)
Conformational Change of Domain III Upon Interaction of Annexin V with Phospholipid Membranes: Excited State Lifetime Distribution	150	(1)
Mobility Change of Trp187 in the Annexin V Membrane Complex: Time-Resolved Fluorescence Anisotropy Study	150	(4)
Accessibility of Trp187 to Acrylamide in the Membrane-Bound Protein	154	(1)
The Interaction of Annexin V with Reverse Micelles	154	(1)
Modification of the Trp187 Environment in Reverse Micelles: Steady-State Fluorescence Emission Spectrum	155	(1)
Excited State Lifetime Distribution of Trp187: Conformational Change in Reverse Micelles	156	(1)
Time-Resolved Fluorescence Anisotropy Decays	157	(1)
Secondary Structure of Annexin V in Reverse Micelles: Circular Dichroism	158	(1)
Discussion	158	(17)
The Role of the Conformational Change of Domain III in the Annexin/Membrane Interactions: Is the Swinging out of Trp187 Crucial for Binding?	161	(2)
The Location of Trp187 at the Membrane/Protein/Water Interface	163	(2)
The Mechanism of the Conformational Change on the Membrane Surface	165	(1)
What could be the Role of the Conformational Change of Domain III of Annexin V in the Formation of the Trimeric Complexes at the Membrane Surface	166	(1)
References	167	(8)
Tryptophan Calmodulin Mutants
Jacques Haiech
Marie-Claude Kilhoffer
Introduction	175	(3)
Building Tryptophan Containing Calmodulin Mutants	178	(5)
Where to Insert the Tryptophanyl Residue?	179	(1)
How to Insert Tryptophan?	180	(1)
Expression, Purification and Characterization of the Tryptophan Containing Mutants	180	(3)
Analysis of the Tryptophan Containing Calmodulin Mutants	183	(1)
The Mutants Have To Be Isostructural	183	(1)
The Mutants Have To Be Similar to SynCaM in their Calcium Binding Properties	183	(1)
Using Tryptophan Containing Calmodulin Mutants as a Tool to Obtain Deeper Insight Into the Structure and Calcium Binding Mechanism of Calmodulin	184	(16)
Fluorescent Properties of the Tryptophan Containing SynCaM Mutants	185	(4)
Calcium Titration of the Mutants: A Probe of the Sequential Ca2+ Binding Mechanism	189	(1)
Ca2+ Titrations in the Absence of Ethylene Glycol	189	(2)
Ca2+ Titrations in the Presence of Ethylene Glycol	191	(1)
Comments	192	(1)
Fluorescence Stopped-Flow as a Probe of a Limiting Step in the Kinetics of Ca2+ Binding to Calmodulin	193	(1)
Fluorescence Lifetimes of Tryptophan Mutants	194	(1)
Time Domain Lifetimes	194	(2)
Time resolved Spectra: A Probe of the Selection of Conformation Upon Calcium Binding	196	(2)
Measurements of Distances by Radiationless Energy Transfer	198	(2)
Perspectives and Open Questions	200	(11)
References	201	(10)
Luminescence Studies with Trp Aporepressor and Its Single Tryptophan Mutants
Maurice R. Eftink
Introduction	211	(1)
Fluorescence Studies with Wild Type and Mutant Forms of Trp Aporepressor	212	(6)
Summary	218	(3)
References	219	(2)
Heme-Protein Fluorescence
Rhoda Elison Hirsch
Introduction	221	(1)
Techniques to Detect Heme-Protein Fluorescence	222	(3)
Origin and Assignment of the Steady-State Fluorescence Signal	225	(17)
Intrinsic Fluorescence	227	(1)
Apoglobins	228	(1)
Steady-State Fluorescence of Intact Heme-Proteins	228	(5)
Coupling of Diverse Spectroscopic Approaches Confirms Fluorescence Assignments	233	(1)
Time-Resolved Intrinsic Fluorescence Studies of Heme-Proteins Reveals Complex Data, But Data That is Consistent with Known Protein Trp Fluorescence	234	(1)
Interpretations of the Multiexponential Decays Remains Unresolved	235	(7)
Extrinsic Fluorescence Probing	242	(3)
Quenching of Extrinsic Fluorescence Upon Binding by Heme or Heme-Proteins	245	(1)
Vital Novel Functions of Heme-Proteins Are Now Being Uncovered	246	(11)
References	247	(10)
Conformation of Troponin Subunits and Their Complexes from Striated Muscle
Herbert C. Cheung
Wen-Ji Dong
Introduction	257	(1)
Topography and Structure of Troponin Subunits	258	(3)
Troponin Complex	258	(1)
Troponin	259	(1)
Troponin I and Troponin	260	(1)
Conformation of Skeletal Muscle TnC	261	(8)
Conformation of the Regulatory Domain of Skeletal TnC	261	(1)
Properties of Single-Tryptophan TnC Mutants	262	(1)
Structure and Fluorescence of Mutant F22W	262	(2)
Fluorescence of Other Single-Tryptophan Mutants	264	(1)
Conformational.Change Induced By Activator Ca2+	265	(4)
The N-Domain Conformation of Cardia Muscle TnC	269	(4)
Comparison of Cardiac TnC and Skeletal TnC Conformation	273	(1)
Topography of Cardiac Troponin	274	(6)
FRET Studies of Cardiac TnI	274	(1)
The General Shape of cTnI	274	(1)
The CTnC-cTnI Complex	275	(5)
Summary and Prospects	280	(5)
References	281	(4)
Fluorescence of Extreme Thermophilic Proteins
Sabato D'Auria
Mose Rossi
Ignacy Gryczynski
Joseph R. Lakowicz
Introduction	285	(1)
Thermophilic Micro-Organisms	286	(1)
Thermophilic Enzymes	287	(2)
Conformational Stability of Extreme Thermophilic Enzymes	289	(3)
Inter-Relationships of Enzyme Stability-Flexibility-Activity	292	(1)
Hyperthermophilic β-glycosidase from the Archaeon S. solfatricus	293	(2)
Effect of Temperature on Tryptophanyl Emission Decay	295	(5)
Effect of pH on Tryptophanyl Emission Decay of Sβgly	300	(1)
Effect of Organic Solvents on Sßgly Tryptophanyl Emission Decay	300	(7)
Acknowledgments	303	(1)
References	303	(4)
Index	307