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Dipolar Couplings for Structure Refinement 301 23 The Use of Dipolar Couplings for the Structure Refinement of a Pair of Calcium-Binding EGF Domains Jonathan Boyd, Iain D. Campbell, and A. Kristina Downing 1. Introduction The calcium-binding (cb) epidermal growth factor-like (EGF) domain is a common variant of the EGF module that contains a consensus sequence associated with ligation of a single calcium ion (1–3). Many extracellular proteins include cbEGF domains, and several of these have been associated with human disease (reviewed in ref. 4). Two examples from this group are human fibrillin-1 and the low-density liproprotein (LDL) receptor. Moreover, mutations within these proteins have been linked to the Marfan syndrome and familial hypercholesterolaemia, respectively. Recently, we have been using solution NMR to probe the structure, calcium-binding properties and dynamics of tandem cbEGF domains from these two proteins. The structure of the thirty-second and thirty-third cbEGF domains from human fibrillin-1 (cbEGF32–33) revealed that, in the presence of calcium ions, the two modules adopt a stable rigid rod-like conformation (5). Analysis of the structure suggests that the domain arrangement is stabilized by the calcium binding to the C-terminal domain in addition to interdomain hydrophobic packing interactions. Furthermore, protein sequence analysis has predicted that the relative orientation of these domains might be a structurally conserved feature of a number of functionally distinct proteins. To test this hypothesis, we have probed the specificity of cbEGF domain packing interactions via investigation of the structure of the cbEGF pair from the LDL receptor (LDLR-AB) (Downing, A. K., et al., manuscript in preparation). For the structure determination of this module pair, we have measured and implemented methods that 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 301
302 Boyd et al. utilize residual dipolar coupling-derived restraints (6–8), in addition to employing the usual NMR conformational restraints derived from 1 H– 1 H homonuclear nuclear Overhauser enhancements (NOE), 3 J Hα–HN dihedral angles and slow NH exchange. The use of residual dipolar couplings for structure determination has recently been reviewed (9). For this chapter, we confine our discussion to a consideration of the dipolar couplings arising between the amide 15 N nucleus and its directly attached proton from the peptide backbone. In a rigid molecule, the magnitude of the dipole–dipole coupling between the magnetic moments of the 1 H and 15 N nuclei depends upon the orientation of the static magnetic field B 0 relative to the internuclear vector. It is well established that under normal solution conditions, molecules tumble isotropically, sampling all orientations of conformational space equally. Under these conditions, the dipolar coupling is averaged to zero and does not contribute to the resonance frequencies (although it does strongly influence the relaxation parameters T 1, T 2 and the 1 H– 15 N NOE). However, in a major development, Tjandra and Bax (10) showed that a relatively small net sample orientation could be introduced when employing a solution mixture containing, in addition to the protein, a completely magnetically oriented nematic uniaxial liquid crystalline phase, known as bicelles (11,12). More recently, it has been shown that phage or purple membrane may also be used to induce tunable alignment (13–15). A typical experimental protocol is to adjust the concentration of alignment agent to achieve a net sample orientation of about 10 –3 for the protein. The spectra from the protein then still retain the relative simplicity found for an isotropic solution, with the dipolar coupling reduced from the maximum static molecule value of about 21.6 KHz for an isolated 15 N– 1 H spin pair to a manageable range of a few tens of Hz. NMR structure determination methodology usually relies on identifying, from multidimensional 1 H– 1 H NOESY spectra, many local interatomic interactions that are converted to different classes of distance restraint (up to about 5Å) depending on the intensities of the assigned crosspeaks (16). Because of the time-averaged nature of NMR data and other sources of inaccuracy such as peak overlap and spin diffusion, there is a tendency for long range error propagation because the structure determination uses predominantly short range 1 H– 1 H distance restraints. There have been attempts to reduce the effect of spin diffusion by sample deuteration. It has been demonstrated that distance restraints can be derived from backbone amide protons separated by up to about 7 Å, in a deuterated protein (17). In contrast to short-range NOEs, residual dipolar coupling restraints are related to a global principal axis system. In a dilute solution of uniaxial magnetically oriented bicelles the protein molecules still tumble rapidly, but with
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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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Page 271 and 272: 252 Weljie and Heringa 44. Thompson
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Page 275 and 276: 256 Li et al. In order for these ex
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Page 287 and 288: 268 Mal et al. NMR data, X-PLOR (11
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Page 291 and 292: 272 Mal et al. 3.1.2.1. AMBIGUOUS D
Page 293 and 294: 274 Mal et al. molecular dynamics a
Page 295 and 296: 276 Mal et al. Assuming a rigid bod
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Page 299 and 300: 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 315 and 316: 296 Werner et al. Table 2 Models of
Page 317 and 318: 298 Werner et al. 9. Reinhardt, D.
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Page 323 and 324: 304 Boyd et al. Fig. 1. (A) The sol
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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
Page 343 and 344: 324 Yap et al. 2. Ikura, M. (1995)
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 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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352 Walsh et al. Fig. 6. CaM-depend
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354 Walsh et al. 3. Cho, M. J., Vag
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356 Hughes et al. The electroporati
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358 Hughes et al. 4. 1 M Na 2CO 3.
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360 Hughes et al. range, repeat ste
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362 Hughes et al. Table 1 Examples
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366 Persechini of the different CaM
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368 Persechini Fig. 1. Schematic re
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370 Persechini 2. 1 L of Terrific b
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372 Persechini Fig. 4. Titration wi
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374 Persechini Table 1 Parameters f
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376 Persechini Fig. 6. The [Ca 2+ -
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378 Persechini determined if the di
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380 Persechini relatively insensiti
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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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