Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Enzymatic Assays 339 26 Enzymatic Assays to Compare Calmodulin Isoforms, Mutants, and Chimeras Michael P. Walsh, Jacquelyn E. Van Lierop, Cindy Sutherland, Ritsu Kondo, and J. David Johnson 1. Introduction Calmodulin (CaM), the principal protein mediator of cellular Ca 2+ signals, interacts with some 80 target proteins, many of which are enzymes that are activated by CaM in a Ca 2+ -dependent manner. Mammalian genomes contain at least three differentially regulated CaM genes that encode the same protein (1). On the other hand, multiple genes encode several CaM isoforms in plants. For example, the soybean genome contains at least five CaM genes that encode four distinct isoforms (2). Studies of CaM chimeras, mutants, and isoforms indicate that the interactions of CaM with different target proteins and the molecular mechanisms of activation of CaM-dependent enzymes vary depending on the target enzyme. Thus, for example, the soybean CaM isoform SCaM-1 (90.5% identical in sequence to human CaM) activates calcineurin (CaN; type 2B protein serine/ threonine phosphatase) but SCaM-4 (77% sequence identity to human CaM) does not; on the other hand, SCaM-4 activates nitric oxide synthase (NOS) but SCaM-1 does not (3). The fact that SCaM-4 acts as a competitive inhibitor of SCaM-1-mediated activation of CaN and SCaM-1 acts as a competitive inhibitor of SCaM-4-mediated activation of NOS indicates that both plant isoforms bind to the same site on the target enzyme, but in one case (different for each target enzyme) binding is not coupled to activation. Several instances have been described of site-specific mutations in CaM resulting in loss of activation of a specific target enzyme with little effect on binding. For example, replacement of M144 by V in mammalian CaM has no effect on activation of Ca 2+ /CaMdependent cyclic nucleotide 3':5'-phosphodiesterase (PDE) or CaN, but con- 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 339
340 Walsh et al. verts CaM from an activator to a competitive antagonist of NOS (4). A great deal can be learned, therefore, from studying the effect of CaM mutants and isoforms on the activation of various CaM target enzymes. In this chapter, we describe assay methods for such a comparison using five CaM target enzymes: PDE, CaN, NOS, myosin light chain kinase (MLCK), and Ca 2+ /CaM-dependent protein kinase II (CaM kinase II). CaM-dependent PDE catalyzes the hydrolysis of cAMP and cGMP to the corresponding 5'-nucleoside monophosphates, thereby terminating cyclic nucleotide signaling (5). This enzyme, therefore, represents an important point of cross-talk between Ca 2+ and cyclic nucleotide signaling pathways. CaN is a Ca 2+ /CaM-dependent protein serine/threonine phosphatase with a relatively narrow substrate specificity (6). It has diverse regulatory roles, e.g., T-lymphocyte activation, regulation of neurotransmitter release, and modulation of long-term changes in synaptic plasticity. It is the target of the immunosuppressive drugs, FK506 and cyclosporin A. NOS catalyzes the formation of the intercellular messenger nitric oxide (NO) from L-arginine. NO is a major regulator in the nervous, immune, and cardiovascular systems (7). There are two classes of NOS: constitutive and inducible. Constitutive NOS is regulated by Ca 2+ -dependent interaction with CaM, whereas inducible NOS contains tightly bound CaM that is not dissociated by chelation of Ca 2+ ions. MLCK plays a key role in the regulation of smooth muscle contraction and nonmuscle motility via the specific phosphorylation of myosin II (8). Finally, CaM kinase II, unlike MLCK, has a large number of substrates and is, therefore, involved in the regulation of diverse physiological processes including synaptic transmission, secretion, and gene expression (9). For activation of PDE, CaN, and NOS by CaM, we describe continuous assays that allow enzymatic activity to be monitored by changes in fluorescence or absorption upon enzyme activation. For activation of MLCK, CaM kinase II, and NOS by CaM, we describe radioisotope-based assays that follow incorporation of 32 P from [γ- 32 P]ATP into the 20-kDa light chain of myosin II (LC 20), incorporation of 32 P from [γ- 32 P]ATP into caldesmon and conversion of L-[ 14 C] arginine to L-citrulline and NO, respectively. For most of these enzymes, CaM binds and removes a pseudosubstrate (autoinhibitory) domain from the enzyme’s active site resulting in enzyme activation. CaM activation of NOS is more complicated (10,11). CaM binds NOS between its N-terminal oxygenase domain and its C-terminal reductase domain and it stimulates NADPH oxidation and reduction of bound flavin in NOS’s reductase domain (11). CaM also facilitates electron transfer from the reductase domain to the heme-containing oxygenase domain, resulting in the conversion of L-Arg to NO and L-citrulline. Finally, CaM can stimulate the intermolecular transfer of electrons from NOS’s reductase domain to exogenous electron acceptors like cytochrome c (cyt c). Thus, for activation of NOS
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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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Page 317 and 318: 298 Werner et al. 9. Reinhardt, D.
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Page 321 and 322: 302 Boyd et al. utilize residual di
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Page 329 and 330: 310 Boyd et al. total and NOE energ
Page 331 and 332: 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
Page 341 and 342: 322 Table 1 Angle and Distance Outp
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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
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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 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
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390 Török et al. Fig. 3. Peptides
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392 Török et al. Table 1 Peptide
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394 Török et al. Fig. 7. Nanospra
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396 Török et al. Fig. 9. Nanospra
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398 Török et al. Fig. 11. Electro
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400 Török et al. 3 µM FL-calmodu
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402 Török et al. Fig. 14. Localiz
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404 Török et al. Fig. 16. Indicat
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406 Török et al. the significantl
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408 Török et al.
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410 Index substitutes, see Fluoresc
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412 Index Stern-Volmer plot, 78, 81
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414 Index Phenothiazines, see Calmo
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