Contents
1. Intrinsic Fluorescence of Proteins
Maurice R. Eftink
1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3. Patterns in Protein Fluorescence . . . . . . . . . . . . . . . . . . . . . .
1.4. Some Recent Topics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.5. Open Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Spectral Enhancement of Proteins by in vivo Incorporation of
Tryptophan Analogues
J. B. Alexander Ross, Elena Rusinova, Linda A. Luck, and
Kenneth W. Rousslang
2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1. Brief History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. In vivo Analogue Incorporation
......................
2.2.1. A General Approach for in vivo Incorporation
of Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Analyzing the Efficiency of Analogue
Incorporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Spectral Features of TRP Analogues . . . . . . . . . . . . . . . . . .
2.3.1. Absorption of Analogues . . . . . . . . . . . . . . . . . . . . . .
2.3.2. Fluorescence- Analogue Models . . . . . . . . . . . . . . . . .
2.3.3. Fluorescence-Analogue Containing Proteins . . . . . . .
2.3.4. Phosphorescence- Analogue Models . . . . . . . . . . . . . .
2.3.5. Phosphorescence A
- nalogue Containing
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1
2
4
9
12
13
13
17
19
21
23
26
29
30
31
33
34
36
37
39
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Contents
3. Room Temperature Tryptophan Phosphorescence as a Probe of
Structural and Dynamic Properties of Proteins
Vinod Subramaniam, Duncan G. Steel, and Ari Gafni
3.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Factors Influencing Tryptophan Phosphorescence in
Fluid Solution and in Proteins . . . . . . . . . . . . . . . . . . . . . . .
3.3. Protein Dynamics and Folding Studied Using RTP . . . . . . .
3.3.1. Alkaline Phosphatase . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2. Azurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3. Beta-Iactoglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4. Ribonuclease T1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. New Developments in RTP for Protein Studies . . . . . . . . . .
3.4.1. Distance Measurements using RTP (Diffusion
enhanced energy transfer, electron transfer and
exchange interactions) . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.2. H-D Exchange Studies . . . . . . . . . . . . . . . . . . . . . . . .
3.4.3. Circularly Polarized Phosphorescence (CPP) . . . . . . .
3.4.4. Stopped Flow RTP . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4.5. RTP from trp Analogues . . . . . . . . . . . . . . . . . . . . . .
3.4.6. Concluding Remarks and Prospects for the
Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
45
48
48
51
51
52
53
53
55
55
58
58
59
60
4. Azurins and Their Site-Directed Mutants
Giampiero Mei, Nicola Rosato, and Alessandro Finazzi Agriο
∨
4.1. A Brief Overview on Azurin and its Dynamic
Fluorescence Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Copper-Containing Azurins . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. The Apo-Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
67
70
71
75
79
79
5. Barnase: Fluorescence Analysis of a Three Tryptophan Protein
Yves Engelborghs and Alan Fersht
5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Results Obtained by the Method of Subtraction . . . . . . . . .
5.2.1. pH-Dependency of the Fluorescence . . . . . . . . . . . . .
83
85
85
Contents
xv
5.2.2.
5.2.3.
5.2.4.
5.2.5.
The Effect of Removing W35 . . . . . . . . . . . . . . . . . . .
The Effect of Removing W71 . . . . . . . . . . . . . . . . . . .
The Effect of Removing W94 . . . . . . . . . . . . . . . . . . .
Calculation of the Absorption and Fluorescence
Emission Spectra of the Individual
Tryptophans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.6. Calculations of the Forster Energy-Transfer
on the Basis of Spectral Data . . . . . . . . . . . . . . . . . .
5.2.7. The Fluorescence Lifetimes . . . . . . . . . . . . . . . . . . . .
5.2.7.1. Measured and Calculated Lifetimes . . . . . . .
5.2.7.2. Energy Transfer Calculations Using
Lifetime Data . . . . . . . . . . . . . . . . . . . . . . . .
5.2.8. Discussion of Data Obtained from Single
Tryptophan Mutants . . . . . . . . . . . . . . . . . . . . . . . . . .
Characterization of the Double Mutant Protein . . . . . . . . .
5.3.1. Steady-State Fluorescence Parameters . . . . . . . . . . .
5.3.2. Fluorescence Lifetimes . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3. Calculation of the Fluorescence Decay
Parameters of Multi-Tryptophan Proteins
from the Emission of Single-Tryptophan
Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluorescence Anisotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Steady-State Phosphorescence . . . . . . . . . . . . . . . . . . . . . . . .
Concentration Dependence of Phosphorescence
Intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
97
99
100
6. Fluorescence Study of the DsbA Protein from Escherichia Coli
Alain Sillen, Jens Hennecke, Rudi Glockshuber, and
Yves Engelborghs
6.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Fluorescence Properties of W76 . . . . . . . . . . . . . . . . . . . . . .
6.3. Fluorescence Properties of W 126 . . . . . . . . . . . . . . . . . . . . .
6.3.1. Quenching Analysis . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.2. Molecular Mechanics . . . . . . . . . . . . . . . . . . . . . . . . .
6.3.3. Linking the Conformations with the Lifetimes . . . . .
6.4. Overall Scheme of the Quenching in DBSA
............
6.5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
103
106
112
112
114
114
115
115
119
5.3.
5.4.
5.5.
5.6.
5.7.
85
86
86
87
88
89
89
91
92
93
93
94
95
96
97
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Contents
7. The Conformational Flexibility of Domain III of Annexin V is
Modulated by Calcium, pH and Binding to Membrane/
Water Interfaces
Jaques Gallay, Jana Sopkova, and Michael Vincent
7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2. Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.1. Protein Preparation and Chemicals . . . . . . . . . . . . . .
7.2.2. Preparation of Phospholipidic Vescicles and
Reverse Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.3. Steady-State Fluorescence Measurements . . . . . . . . .
7.2.4. Time-Resolved Fluorescence Measurements . . . . . . .
7.2.5. Analysis of the Time-Resolved Fluorescence Data . .
7.2.5.1. Fluorescence Polarized Fluorescence
Intensity Decays . . . . . . . . . . . . . . . . . . . . . .
7.2.5.2. Excited State Lifetime Distribution . . . . . . .
7.2.5.3. Rotational Correlation Time
Distribution . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.5.4. Wobbling-in-Cone Angle Calculation . . . . .
7.2.6. Absorbance and Circular Dichroism
Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.1. Effect of Calcium on the Structure and Dynamics
of Domain III of Annexin V . . . . . . . . . . . . . . . . . . .
7.3.1.1. UV- Difference Absorption Spectra . . . . . . .
7.3.1.2. Circular Dichroism . . . . . . . . . . . . . . . . . . . .
7.3.1.3. Steady-State Fluorescence of Trp187 . . . . . .
7.3.1.4. Time-Resolved Fluorescence Intensity
Decay of Trp187 . . . . . . . . . . . . . . . . . . . . . .
7.3.1.5. Fluorescence Anisotropy of Trp187 . . . . . . .
7.3.2. Effect of pH on the Conformation and
Dynamics of Domain III of Annexin V . . . . . . . . . .
7.3.2.1. Steady-State Fluorescence Emission
Spectrum of Trp187 . . . . . . . . . . . . . . . . . . .
7.3.2.2. Excited State Lifetime Heterogeneity of
Trp187 at Different pH . . . . . . . . . . . . . . . . .
7.3.2.3. Time-Resolved Fluorescence Anisotropy
Study as a Function of pH . . . . . . . . . . . . . .
7.3.2.4. Accessibility of Trp187 to Acrylamide,
a Water Soluble Fluorescence Quencher . . .
7.3.2.5. Secondary Structure of Annexin V as a
Function of pH: Circular Dichroism
Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
125
125
125
126
126
127
127
128
129
130
131
132
132
132
132
135
137
139
143
143
144
145
146
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Contents
7.3.3. The Interaction of Annexin V with Small
Unilamellar Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.3.1. Polarity Change Around Trp187
Induced by the Interaction with
Membranes: Steady-State Fluorescence
Spectra of Trp187 . . . . . . . . . . . . . . . . . . . . .
7.3.3.2. Conformational Change of Domain III
Upon Interaction of Annexin V with
Phospholipid Membranes: Excited State
Lifetime Distribution . . . . . . . . . . . . . . . . . .
7.3.3.3. Mobility Change of Trp187 in the
Annexin V Membrane Complex:
Time-Resolved Fluorescence Anisotropy
Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.3.4. Accessibility of Trp187 to Acrylamide
in the Membrane-Bound Protein . . . . . . . . .
7.3.4. The Interaction of Annexin V with Reverse
Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.4.1. Modification of the Trp187
Environment in Reverse Micelles:
Steady-State Fluorescence Emission
Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.4.2. Excited State Lifetime Distribution of
Trp187: Conformational Change in
Reverse Micelles . . . . . . . . . . . . . . . . . . . . . .
7.3.4.3. Time-Resolved Fluorescence Anisotropy
Decays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.4.4. Secondary Structure of Annexin V in
Reverse Micelles: Circular Dichroism . . . . .
7.4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4.1. The Role of the Conformational Change of
Domain III in the Annexin/Membrane
Interactions: Is the Swinging out of Trp187
Crucial for Binding? . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4.2. The Location of Trp187 at the Membrane/
Protein/Water Interface . . . . . . . . . . . . . . . . . . . . . . .
7.4.3. The Mechanism of the Conformational Change
on the Membrane Surface . . . . . . . . . . . . . . . . . . . . .
7.4.4. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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149
149
150
151
154
154
155
156
157
158
158
161
163
165
166
167
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Contents
8. Tryptophan Calmodulin Mutants
Jacques Haiech and Marie-Claude Kilhoffer
8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2. Building Tryptophan Containing Calmodulin Mutants . . . .
8.2.1. Where to Insert the Tryptophanyl Residue? . . . . . . .
8.2.2. How to Insert Tryptophan? . . . . . . . . . . . . . . . . . . . .
8.2.3. Expression, Purification and Characterization of
the Tryptophan Containing Mutants . . . . . . . . . . . .
8.3. Analysis of the Tryptophan Containing Calmodulin
Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.1. The Mutants Have To Be Isostructural . . . . . . . . . . .
8.3.2. The Mutants Have To Be Similar to SynCaM
in their Calcium Binding Properties . . . . . . . . . . . . .
8.4. Using Tryptophan Containing Calmodulin Mutants as a
Tool to Obtain Deeper Insight Into the Structure and
Calcium Binding Mechanism of Calmodulin . . . . . . . . . . .
8.4.1. Fluorescent Properties of the Tryptophan
Containing SynCaM Mutants . . . . . . . . . . . . . . . . . .
8.4.2. Calcium Titration of the Mutants: A Probe of the
Sequential Ca2+ Binding Mechanism . . . . . . . . . . . . .
8.4.2.1. Ca 2+ Titrations in the Absence of
Ethylene Glycol . . . . . . . . . . . . . . . . . . . . . . .
8.4.2.2. Ca2+ Titrations in the Presence of
Ethylene Glycol . . . . . . . . . . . . . . . . . . . . . . .
8.4.2.3. Comments . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.4.2.4. Fluorescence Stopped-Flow as a Probe
of a Limiting Step in the Kinetics of
Ca2+ Binding to Calmodulin . . . . . . . . . . . . .
8.4.3. Fluorescence Lifetimes of Tryptophan
Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.4.3.1. Time Domain Lifetimes . . . . . . . . . . . . . . . .
8.4.3.2. Time resolved Spectra: A Probe of the
Selection of Conformation Upon
Calcium Binding . . . . . . . . . . . . . . . . . . . . . .
8.4.4. Measurements of Distances by Radiationless
Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5. Perspectives and Open Questions . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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178
179
180
180
183
183
183
184
185
189
189
191
192
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194
194
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198
200
201
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9. Luminescence Studies with Trp Aporepressor and Its Single
Tryptophan Mutants
Maurice R. Eftink
9.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2. Fluorescence Studies with Wild Type and Mutant Forms
of Trp Aporepressor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10. Heme-Protein Fluorescence
Rhoda Elison Hirsch
10.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2. Techniques to Detect Heme-Protein Fluorescence . . . . . .
10.3. Origin and Assignment of the Steady-State Fluorescence
Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.1. Intrinsic Fluorescence . . . . . . . . . . . . . . . . . . . . . .
10.3.2. Apoglobins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.3. Steady-State Fluorescence of Intact
Heme-Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.4. Coupling of Diverse Spectroscopic Approaches
Confirms Fluorescence Assignments . . . . . . . . . .
10.3.5. Time-Resolved Intrinsic Fluorescence Studies of
Heme-Proteins Reveals Complex Data, But Data
That Is Consistent with Known Protein Trp
Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3.5.1. Interpretations of the Multiexponential
Decays Remains Unresolved . . . . . . . . .
10.4. Extrinsic Fluorescence Probing
10.5. Quenching of Extrinsic Fluorescence Upon Binding by
Heme or Heme-Proteins . . . . . . . . . . . . . . . . . . . . . . . . . .
10.6. Vital Novel Functions of Heme-Proteins Are Now Being
Uncovered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211
212
218
219
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.
221
222
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225
227
228
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228
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233
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234
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242
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245
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246
247
11. Conformation of Troponin Subunits and Their Complexes from
Striated Muscle
Herbert C. Cheung and Wen-Ji Dong
11.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2. Topography and Structure of Troponin Subunits . . . . . . . .
257
258
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Contents
11.3.
11.4.
11.5.
11.6.
11.7.
11.2.1. Troponin Complex . . . . . . . . . . . . . . . . . . . . . . . . .
11.2.2. Troponin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2.3. Troponin I and Troponin T . . . . . . . . . . . . . . . . . .
Conformation of Skeletal Muscle TnC . . . . . . . . . . . . . . .
11.3.1. Conformation of the Regulatory Domain of
Skeletal TnC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.2. Properties of Single-Tryptophan
TnC Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.2.1. Structure and Fluorescence of Mutant
F22W . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.2.2. Fluorescence of Other
Single-Tryptophan Mutants . . . . . . . . . .
11.3.2.3. Conformational Change Induced By
Activator Ca2+ . . . . . . . . . . . . . . . . . . . . .
The N-Domain Conformation of Cardia Muscle TnC
...
Comparison of Cardiac TnC and Skeletal TnC
Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Topography of Cardiac Troponin . . . . . . . . . . . . . . . . . . . .
11.6.1. FRET Studies of Cardiac TnI . . . . . . . . . . . . . . . .
11.6.2. The General Shape of cTnI . . . . . . . . . . . . . . . . . .
11.6.3. The cTnC-cTnI Complex . . . . . . . . . . . . . . . . . . . .
Summary and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
258
259
260
261
261
262
262
264
265
269
273
274
274
274
275
280
281
12. Fluorescence of Extreme Thermophilic Proteins
Sabato D’Auria, Mose Rossi, Ignacy Gryczynski, and
Joseph R . Lakowicz
12.1.
12.2.
12.3.
12.4.
12.5
12.6
12.7.
12.8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermophilic Micro-Organisms . . . . . . . . . . . . . . . . . . . . .
Thermophilic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conformational Stability of Extreme Thermophilic
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Inter-Relationships of Enzyme Stability-FlexibilityActivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hyperthermophilic β-glycosidase from the Archaeon
S. solfataricus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of Temperature on Tryptophanyl Emission
Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of pH on Tryptophanyl Emission Decay of
S βgly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
285
286
287
289
292
293
295
300
Contents
xxi
12.9. Effect of Organic Solvents on Sβgly Tryptophanyl
Emission Decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
300
303
303
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
307