Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Spatial Distribution of Ca 2+ -Binding Proteins 383 29 Studying the Spatial Distribution of Ca 2+ -Binding Proteins How Does it Work for Calmodulin? Katalin Török, Richard Thorogate, and Steven Howell 1. Introduction Calmodulin is a ubiquitous Ca 2+ -switch protein whose in vitro properties have been widely studied (1). Visualization of calmodulin levels and functional changes in living cells allows investigations of how calmodulin is involved in organizing specific cellular responses to various stimuli. The advancement of several protein chemistry, biochemical, and microscopic techniques has made the direct study of calmodulin in cellular function less perturbing, more sensitive, and of higher temporal and spatial resolution. For example, more selective fluorescent-labeling techniques directed at strategically positioned Lys and Cys residues in the protein are now available, the latter are introduced by site-directed mutagenesis. In addition, the conjugation of calmodulin c-DNA with enhanced green fluorescent protein (GFP) provides increased sensitivity. Taken together, these advances allow the fluorescence signal of the protein to act as an intracellular reporter group of concentration changes in cell compartments, as well as other well-defined molecular events (e.g., Ca 2+ and target binding, conformational change). Brighter fluorophores provide increased sensitivity and thus the fluorescent protein may be applied at a lower concentration to act as a tracer of endogenous calmodulin. Calmodulins with probes attached at a single site that have been characterized functionally by comparison to unmodified calmodulin, facilitate clearer data interpretation. Laser-scanning confocal microscopy offers higher resolution so events in living cells can be monitored in greater detail in order to understand calmodulin and its inter- From: Methods in Molecular Biology, vol. 173: Calcium-Binding Protein Protocols, Vol. 2: Methods and Techniques Edited by: H. J. Vogel © Humana Press Inc., Totowa, NJ 383
384 Török et al. actions in the cell. When interpreting the fluorescent signals, it has to be considered that both the concentration and the liganded state of calmodulin may change simultaneously. Vertebrate calmodulins can be modified most readily on lysine residues. Several nonfluorescent lysine reagents have been attached to calmodulin and, in most cases, the reagents predominantly modified Lys 75 (2). The higher reactivity of Lys 75 as opposed to the other lysine residues in the protein, originates in its lower pK a value as determined by NMR spectroscopy (3). Here, we have used the isomeric fluorescein dichlorotriazine, 5-DTAF, which readily modifies amines in proteins. In this chapter, we use the example of Lys 75-labeled FL-calmodulin, which has already been used for imaging in living cells. We intend to provide a comprehensive approach including the synthesis and analysis of the fluorescent calmodulin and its application into living cells. We close with demonstrating how the activation state of calmodulin can be monitored in living cells by confocal imaging. 2. Materials 2.1. Synthesis of FL-Calmodulin 1. 4 mM 5-DTAF (D-16, Molecular Probes), stock solution dissolved in dimethyl formamide (DMF). 2. 60 µM pig brain calmodulin (4) in 200 mM Tris-HCl (pH 8.5) containing 20 mM CaCl2. 3. PD10 Columns (Pharmacia Biotech). 4. Solvent A: 0.1% solution trifluoroacetic acid (TFA) (HPLC grade)/H2O. 5. Solvent B: 0.082% TFA solution/acetonitrile (HPLC grade). 6. Semipreparative Vydac C18 reverse-phase column 10 × 250 mm (Hichrom Ltd., UK). 7. 1 M Tris-HCl buffer, pH 7.5. 2.2. Characterization of Lys 75 Singly Labeled FL-Calmodulin 1. Freeze dried, desalted, singly labeled Lys 75 FL-calmodulin 2. Digestion mixture containing 100 mM NH 4HCO 3, pH 9.0, 2 mM EGTA, and 10 µg/mL trypsin, TPCK treated (Sigma, Aldrich, UK). 3. Solvent A: 0.1% solution trifluoroacetic acid (TFA) (HPLC grade)/H 2O. 4. Solvent B: 0.082% TFA solution/acetonitrile (HPLC grade). 5. Semipreparative Vydac C18 reverse-phase column 10 × 250 mm (Hichrom Ltd.). 6. Electrospray mass spectrometer. 2.3. Other NH 2-Reactive Fluorescent Probes 1. Dansyl chloride. 2. TA-Cl [2,4-dichloro-6-(4-N,N-diethylaminophenyl)-1,3,5-triazine]. 3. Texas Red. 4. Cy5 [N-(N-hydoxy-succinimidyl-carboxypentyl)-N 1 -(ethyl)-indodicarbocyamine-5,5 1 -disulforate].
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Methods in Molecular BiologyTM Biol
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M E T H O D S I N M O L E C U L A R
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© 2002 Humana Press Inc. 999 River
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Preface Calcium plays an important
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Contents Dedication ...............
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Contents xi 26 Enzymatic Assays to
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xiv Contents of Companion Volume 15
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xvi Contributors LESLIE D. HICKS
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20 Dean, Kelsey, and Re
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2 Yazawa
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4 Yazawa Fig. 1. Schematic represen
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6 Yazawa Fig. 2. Shop drawing for t
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8 Yazawa DuPont-NEN and the molar c
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10 Yazawa Fig. 4. Examples of the C
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12 Yazawa 5. Plot the calculated Ca
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14 Yazawa 12. Starovasnik, M. A., D
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16 Fig. 1. Molecular structures and
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18 Linse 7. 0.1 M EDTA. Dissolve 37
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20 Linse iterate the other paramete
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22 Linse tive to small alterations
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24 Linse References 1. Tsien, R. Y.
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26 Haiech and Kilhoffer to use the
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28 Haiech and Kilhoffer where γ is
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30 Haiech and Kilhoffer reporter gr
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32 Haiech and Kilhoffer tein with i
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34 Haiech and Kilhoffer Calmodulin
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36 Haiech and Kilhoffer Fig. 3. Mec
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38 Haiech and Kilhoffer We may sugg
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40 Haiech and Kilhoffer 29. Stewart
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42 Haiech and Kilhoffer 62. Becking
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44 Martin and Bayley Fig. 1. Absorp
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46 Martin and Bayley evaporation. C
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48 Martin and Bayley 4. Set the tem
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50 Martin and Bayley Fig. 2. Near-U
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52 Martin and Bayley discussed by M
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54 Martin and Bayley References 1.
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20 Dean, Kelsey, and Reik
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58 Fabian and Vogel are equipped wi
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60 Fabian and Vogel The penetration
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62 Fabian and Vogel perature, ionic
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64 Fabian and Vogel Fig. 3. IR spec
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66 Fabian and Vogel Fig. 4. Lower t
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68 Fabian and Vogel These correlati
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70 Fabian and Vogel Fig. 5. Amide I
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72 Fabian and Vogel 4. ATR-FTIR spe
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74 Fabian and Vogel 25. Georg, H.,
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76 Weljie and Vogel Fig. 1. Simplif
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78 Weljie and Vogel Fig. 3. Steady-
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80 Weljie and Vogel VIS spectrophot
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82 Weljie and Vogel 4. A series of
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84 Table 1 Advanced Fluorescence Me
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86 Weljie and Vogel References 1. L
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90 Johnson and Tikunova is removed
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92 Johnson and Tikunova function of
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94 Johnson and Tikunova Fig. 2. Rat
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96 Johnson and Tikunova Fig. 3. Rat
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98 Johnson and Tikunova Fig. 4. Ca
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100 Johnson and Tikunova cence inte
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102 Johnson and Tikunova 6. Johnson
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104 Julenius Fig. 1. Under conditio
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106 Julenius 3. Mix 50 µL of NHS s
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108 Julenius Fig. 3. A typical sens
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110 Julenius (2), is used in the Ia
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114 Lopez and Makhatadze Fig. 1. Th
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116 Lopez and Makhatadze 6. Buffers
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118 Lopez and Makhatadze The excess
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122 Lopez and Makhatadze Fig. 1. Is
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124 Lopez and Makhatadze 2.5 mL are
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126 Lopez and Makhatadze pendence o
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128 Hicks et al. equipped with a pu
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130 Hicks et al. Fig. 2. Debye plot
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132 Hicks et al. and 1.0 mg/mL for
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134 Hicks et al. Fig. 3. Sedimentat
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136 Hicks et al. 5. Hayes, D. B. (M
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138 Trewhella and Krueger This chap
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140 Trewhella and Krueger of the sc
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142 Trewhella and Krueger Table 1 C
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144 Trewhella and Krueger beam, or
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146 Trewhella and Krueger At very l
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148 Trewhella and Krueger analytica
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150 Trewhella and Krueger collapsed
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152 Trewhella and Krueger P(r) func
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154 Trewhella and Krueger Fig. 3. S
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156 Trewhella and Krueger concentra
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158 Trewhella and Krueger by a Nati
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162 Doherty-Kirby and Lajoie metal
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164 Doherty-Kirby and Lajoie two Ca
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166 Doherty-Kirby and Lajoie 7. Pep
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168 Doherty-Kirby and Lajoie Fig. 1
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170 Doherty-Kirby and Lajoie Fig. 2
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172 Doherty-Kirby and Lajoie 5. Alt
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174 Doherty-Kirby and Lajoie phosph
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176 Shaw Fig. 1. Ribbon drawing of
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178 Shaw 2.3. Other 1. Chemicals fo
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180 Shaw 3.3.3. Purification 1. Con
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182 Shaw 10. Hodges, R. S., Semchuk
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184 Brokx and Vogel Table 1 An Over
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186 Brokx and Vogel Fig. 1. UV abso
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188 Brokx and Vogel Fig. 2. 20% SDS
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190 Brokx and Vogel it through an a
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192 Brokx and Vogel 8. Weljie, A. M
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196 Berliner Fig. 1. Aqueous X-band
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198 Berliner in the Bruker instrume
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200 Berliner Fig. 3. ESR spectra of
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202 Berliner Fig. 5. Low-temperatur
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204 Berliner 14. Musci, G., Reed, G
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206 Clarke and Vogel cal calcium-bi
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208 Clarke and Vogel Fig. 1. 113 Cd
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214 Clarke and Vogel The linewidth
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218 Drakenberg sands, of Hertz broa
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220 Drakenberg 1. Bloch equations m
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222 Drakenberg Fig. 2. Measurement
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224 Drakenberg Fig. 3. (A) 43 Ca NM
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226 Drakenberg Fig. 5. 43 Ca NMR sp
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228 Drakenberg NMR at sub-mM concen
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230 Drakenberg 30. Shimizu, T., Hat
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232 Weljie and Heringa Table 1 Amin
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234 Weljie and Heringa Table 2 Webs
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236 Weljie and Heringa diction meth
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238 Weljie and Heringa these databa
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240 Weljie and Heringa lineages. Th
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242 Weljie and Heringa compared, an
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244 Weljie and Heringa sequences as
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246 Weljie and Heringa much larger
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248 Weljie and Heringa ment require
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250 Weljie and Heringa 10. Kawasaki
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252 Weljie and Heringa 44. Thompson
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254 Weljie and Heringa
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256 Li et al. In order for these ex
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258 Li et al. 7. Mineral Mixture (s
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260 Li et al. 3. When the 45% D 2O/
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262 Li et al. sion system works eff
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264 Li et al. reliably produce high
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268 Mal et al. NMR data, X-PLOR (11
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270 Mal et al. Table 1 Simulated An
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272 Mal et al. 3.1.2.1. AMBIGUOUS D
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274 Mal et al. molecular dynamics a
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276 Mal et al. Assuming a rigid bod
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278 Mal et al. assign (segid A and
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280 Mal et al. References 1. Drenth
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282 Mal et al. 33. Jeener, J., Meie
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286 Werner et al. Our research has
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288 Fig. 1. (A) T 1, T 2 and the he
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290 Werner et al. tion, using gradi
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292 Werner et al. Fig. 3. (A) Rotat
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294 Werner et al. Fig. 4. (A) Line-
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296 Werner et al. Table 2 Models of
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298 Werner et al. 9. Reinhardt, D.
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300 Werner et al. 39. Press, W. H.,
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302 Boyd et al. utilize residual di
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304 Boyd et al. Fig. 1. (A) The sol
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306 Boyd et al. or significant peak
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308 Boyd et al. Fig. 3. Histogram o
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310 Boyd et al. total and NOE energ
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312 Boyd et al. 2. When studying mu
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314 Boyd et al. 4. Downing, A. K.,
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316 Boyd et al. 35. Schulte-Herbrug
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318 Yap et al. image representation
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320 Yap et al. modified EF-hand is
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322 Table 1 Angle and Distance Outp
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324 Yap et al. 2. Ikura, M. (1995)
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326 Kobayashi that S-100A1 and S-10
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328 Kobayashi 3.2. Coupling of Crom
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330 Kobayashi Fig. 2. Tricine/SDS/P
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Page 359 and 360: 340 Walsh et al. verts CaM from an
Page 361 and 362: 342 Walsh et al. 11. Bovine serum a
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Page 367 and 368: 348 Walsh et al. 3.5. NOS Reduction
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Page 375 and 376: 356 Hughes et al. The electroporati
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Page 379 and 380: 360 Hughes et al. range, repeat ste
Page 381 and 382: 362 Hughes et al. Table 1 Examples
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Page 385 and 386: 366 Persechini of the different CaM
Page 387 and 388: 368 Persechini Fig. 1. Schematic re
Page 389 and 390: 370 Persechini 2. 1 L of Terrific b
Page 391 and 392: 372 Persechini Fig. 4. Titration wi
Page 393 and 394: 374 Persechini Table 1 Parameters f
Page 395 and 396: 376 Persechini Fig. 6. The [Ca 2+ -
Page 397 and 398: 378 Persechini determined if the di
Page 399 and 400: 380 Persechini relatively insensiti
Page 401: 382 Persechini 18. Chafouleas, J. G
Page 405 and 406: 386 Török et al. 3. The reaction
Page 407 and 408: 388 Török et al. Fig. 2. Electros
Page 409 and 410: 390 Török et al. Fig. 3. Peptides
Page 411 and 412: 392 Török et al. Table 1 Peptide
Page 413 and 414: 394 Török et al. Fig. 7. Nanospra
Page 415 and 416: 396 Török et al. Fig. 9. Nanospra
Page 417 and 418: 398 Török et al. Fig. 11. Electro
Page 419 and 420: 400 Török et al. 3 µM FL-calmodu
Page 421 and 422: 402 Török et al. Fig. 14. Localiz
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Page 425 and 426: 406 Török et al. the significantl
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Page 429 and 430: 410 Index substitutes, see Fluoresc
Page 431 and 432: 412 Index Stern-Volmer plot, 78, 81
Page 433 and 434: 414 Index Phenothiazines, see Calmo
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