Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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S-100 <strong>Protein</strong> Interacting Drugs 333 Fig. 4. Affinity chromatography of the recombinant S-100A12 and S-100A13 and Tricine/SDS/PAGE (15) analysis of selected fractions. (A) Elution curves of the recombinant proteins from an Amlexanox-Toyopearl column (O, S-100A12; J, S-100A13). a, Sample loading followed by washing with Ca 2+ -containing buffer (Buffer B); b, addition of the buffer containing 2 mM EGTA (Buffer C); c, addition of the buffer containing 6 M urea (Buffer D). (B) Tricine/SDS/PAGE (12%) analysis of the recombinant S-100A12 and the fractions (4, 15, 40, 44, 60, and 78) from Amlexanox-Toyopearl column chromatography (see A). (C) 12% Tricine/SDS/PAGE analysis of the recombinant S-100A13 and the fractions (4, 15, 25, 26, 55, and 78) from Amlexanox-Toyopearl column chromatography. Size standards (Mw) are shown in kDa.
334 Kobayashi Both recombinant S-100A12 and S-100A13 interacted with the drug in a Ca 2+ -dependent manner and the affinity of S-100A12 to Amlexanox was much higher than that of S-100A13. The observation implies that the spatial arrangement of the amino acid residues that interact with the drug differs in S-100A12 and S-100A13. 3.8.3. Example 2: Fluphenazine-Sepharose Chromatography of Recombinant and Mutant S-100A1 <strong>Protein</strong>s Standardized Fluphenazine-Epoxy activated Sepharose columns of equivalent size and volume (1 mL) as well as fraction size were used to chromatograph equivalent volumes and quantities of recombinant and mutant S-100A1 proteins. Elution profiles and Tricine/SDS/PAGE analyses of the recombinant and mutant S-100A1 proteins are shown in Fig. 5. The recombinant S-100A1 protein bound in the presence of Ca 2+ and was eluted in the presence of EGTA (a Ca 2+ -chelating agent). The C-terminal deletion mutant (∆FFWENS) of S-100A1 was detected in the Ca 2+ buffer wash. These results indicate that C-terminal six residues are necessary for Ca 2+ -dependent interaction of S-100A1 with the drug. Lander et al. (16) and Osterloh et al. (17) demonstrated that proteins lacking the carboxy-terminal nine residues (especially three hydrophobic residues, Phe-88, Phe-89, and Trp-90) of S-100A1 exhibited reduced Ca 2+ -dependent interaction with the Cap Z peptide, TRTK–12. These result indicate that Fluphenazine interacts with S-100A1 at the target recognition domain in the protein. 3.8.4. Example 3: Specificity of Amlexanox-S-100A12 Interaction (Fig. 6) Antiallergic drugs of strikingly heterogeneous chemical structure bind S-100A12 and S-100A13 in a Ca 2+ -dependent manner and can be used for purification of these proteins by a protocol utilizing this Ca 2+ -binding interaction. Ca 2+ -dependent interaction of S-100A1 and S-100B with calmodulin antagonists, such as phenothiazine and W7 was also reported. To analyze specificity of the drug-binding to S-100A12, standardized Fluphenazine-Sepharose and Amlexanox-Toyopearl columns of equivalent size were used. As expected, S-100A12 bound Amlexanox-Toyopearl in the presence of Ca 2+ (Buffer B) and was eluted in the presence of Ca 2+ -chelating agent, EGTA (BufferC). S-100A12 also bound to Fluphenazine- Sepharose in the presence of Ca 2+ and eluted in the presence of EGTA. However, a large amount of the protein was detected in the Ca 2+ buffer wash. The result indicates that S-100A12 has much weaker interaction with Fluphenazine than that with Amlexanox.
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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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Page 307 and 308: 288 Fig. 1. (A) T 1, T 2 and the he
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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
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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
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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
Page 385 and 386: 366 Persechini of the different CaM
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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
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384 Török et al. actions in the c
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386 Török et al. 3. The reaction
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388 Török et al. Fig. 2. Electros
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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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