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Monitoring Ca 2+ -Calmodulin Concentration 365 28 Monitoring the Intracellular Free Ca 2+ -Calmodulin Concentration with Genetically-Encoded Fluorescent Indicator Proteins Anthony Persechini 1. Introduction Calmodulin (CaM) is probably the single most important Ca 2+ -binding protein in the cell by virtue of its central role in converting Ca 2+ signals into biochemical events. It accomplishes this conversion primarily by controlling in a Ca 2+ -dependent manner the activities of a number of different target proteins. Particularly well-studied examples include the myosin light-chain kinases, CaM kinases I, II, and IV, calcineurin, the constitutive nitric oxide synthases, adenylyl cyclases I and VIII, and the cyclic nucleotide phosphodiesterases (1–6). In general, CaM is thought to remain dissociated from its targets at resting free-Ca 2+ concentrations. The protein contains four EF-hand Ca 2+ -binding domains, and it must bind three to four Ca 2+ ions before activating a typical target protein, such as myosin light-chain kinase or phosphodiesterase (7,8). There are several exceptions to this overall picture: proteins containing IQ-motifs, such as neuromodulin or unconventional myosins (9–11), bind Ca 2+ -free CaM as well or better than Ca 2+ -liganded CaM, and CaM is an integral subunit in several proteins, including ryanodine receptors, small conductance potassium channels, inducible nitric oxide synthase, and phosphorylase b kinase (12–15). In spite of these exceptions, it is clear that for many critically important targets, the free concentration of Ca 2+ –CaM ([Ca 2+ –CaM] i) is a crucial determinant of activity. The [Ca 2+ –CaM] i produced at a particular intracellular free-Ca 2+ ion concentration is not easily inferred from in vitro data. It is determined by the amounts and distributions of targets and CaM, the affinities 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 365
366 Persechini of the different CaM-target complexes, and thermodynamic coupling effects on the Ca 2+ -binding affinity of the CaM-target complexes. Furthermore, the amount and subcellular distribution of CaM appears to change during the cell cycle and in response to some stimuli (16–18), and different cell types differ greatly in the total amounts of CaM and targets present (19,20). The distributions of some CaM targets, such as CaM kinase II and calcineurin, also can change dynamically (21,22). Hence, the [Ca 2+ –CaM] i produced at a particular [Ca 2+ ] i is likely to vary among different cell types, and spatiotemporally within cells of the same type. It is clear that a method to monitor [Ca 2+ –CaM] i in intact cells is required before we can investigate transduction of intracellular Ca 2+ signals by CaM. We have developed a family of fluorescent indicators for Ca 2+ –CaM whose responses are based on CaM-dependent changes in fluorescence resonance energy transfer (FRET) between variants of green fluorescent protein (GFP) (23,24). This family of indicators can be stably expressed in mammalian cells, so that [Ca 2+ –CaM] i can be monitored in living cells without the preliminary manipulations needed with organic indicators, such as those typically used to monitor [Ca 2+ ] i. As we have reported elsewhere (25), if CaM is fused to these indicator constructs they become directly responsive to changes in [Ca 2+ ] i. Tsien and co-workers (26) have used a similar approach to develop GFP-based Ca 2+ indicators they term “cameleons.” Whereas GFP-based Ca 2+ indicators clearly have significant utility, they are not the subject of this chapter. We will focus here on the methods used to construct, characterize, and express indicators for Ca 2+ –CaM, and to utilize them to monitor [Ca 2+ –CaM] i in living cells. 2. Materials Materials are generally given within the protocol descriptions, where they can be understood in the context of the procedures in which they are employed. Additional details concerning the materials used are presented here. Unless otherwise stated, all reagents are obtained from standard sources and are of analytical grade. Double-deionized water is used throughout. Equipment and materials required for routine molecular biology procedures are not listed, as these procedures are not detailed in this chapter. 1. Pure CaM: We use calmodulin expressed in Escherichia coli in all our protocols. We have described the procedures for purification of bacterially expressed CaM in detail elsewhere (27). Purified CaM is also commercially available from a number of sources. 2. Sequences encoding EYFP, ECFP, and CaM-binding linkers: DNA sequences encoding the GFP variants are available commercially from Clontech, Inc. (Palo Alto, CA). DNA sequences encoding the CaM-binding linker sequences were
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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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Page 337 and 338: 318 Yap et al. image representation
Page 339 and 340: 320 Yap et al. modified EF-hand is
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Page 345 and 346: 326 Kobayashi that S-100A1 and S-10
Page 347 and 348: 328 Kobayashi 3.2. Coupling of Crom
Page 349 and 350: 330 Kobayashi Fig. 2. Tricine/SDS/P
Page 351 and 352: 332 Kobayashi 0.1% TFA at a flow ra
Page 353 and 354: 334 Kobayashi Both recombinant S-10
Page 355 and 356: 336 Kobayashi Fig. 6. Affinity chro
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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
Page 363 and 364: 344 Walsh et al. Fig. 1. CaM-depend
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 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 and 402: 382 Persechini 18. Chafouleas, J. G
Page 403 and 404: 384 Török et al. actions in the c
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
Page 423 and 424: 404 Török et al. Fig. 16. Indicat
Page 425 and 426: 406 Török et al. the significantl
Page 427 and 428: 408 Török et al.
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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