Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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<strong>Protein</strong> Structure Calculation from NMR 269 3.1. Structure Calculation Methods 3.1.1. X-PLOR NMR data alone are not sufficient to determine the positions of all atoms in a biological macromolecule, but must be supplemented by information about the covalent structure of the protein (the amino acid sequence, bond lengths, bond angles, chiralities, and planar groups) as well as by the steric repulsion between nonbonded atom pairs. Details of structure calculation by X-PLOR are given: 1. Build a protein structure file (psf) from the protein sequence using the standard X-PLOR topology (topallhdg.pro) and peptide bond linkage (toph19.pep). This psf will contain the following information: atom names, types, charges, and masses; residue names and segment names and a list of bond terms, angle terms, dihedral terms, improper terms, explicit hydrogen-bonding terms, explicit nonbonded exclusions, and nonbonded group partitions. It does not contain atomic coordinates, parameters, or restraints. 2. Generate a template structure using standard X-PLOR parameter (parallhdg.pro) and molecular structure (psf) files. The template structure will have an extended conformation with good local geometry and no nonbonded contacts. 3. Create NOE restraints table (see Subheading 4.). 4. Create dihedral angle restraints table (see Subheading 4.). 5. Select an appropriate potential function for NOE restraints. The most commonly used potential function is a flat bottom (square well) potential with a soft asymptote (11). 6. Produce structure from psf with randomized φ and ψ angles. χ i -angles are not affected. 7. Energy minimization using Powell gradient function (50 steps) to remove nonbonded interaction. 8. Molecular dynamics-based simulated annealing (MDSA). Typically, MDSA from an extended template structure with randomized φ and ψ angles works in four stages: a high-temperature search phase, two cooling phases, and a final-energy minimization step. During the high-temperature molecular dynamics (MD) search phase, a low repulsion energy value is used to allow the atoms to pass through each other and to increase the convergence rate. The temperature is reduced from 2000 K to 1000 K in the first cooling phase, and all weights on the different energy terms are gradually brought to their final values (see Table 1). The second cooling phase comprises a slow cooling from 1000 K to 100 K. The final stage comprises 1000 gradient energy minimization steps using the final weighting values of the various energy terms (see Table 1). 3.1.1.1. ANALYSIS OF STRUCTURES DERIVED FROM MDSA 1. Restraints violations: Following calculations, the structures must be analyzed to determine whether they fulfil the given experimental restraints. Usually a viola-
270 Mal et al. Table 1 Simulated Annealing Protocol Stage 1 2 3 4 Temperaturea (K) 2000 2000 → 1000 1000 → 100 100 Number of steps Parameters and force constants 6500 3500 3000 1000 kNOE (kcal mol –1 Å –2 ) 10 → 50 50 50 50 krepel (kcal mol –1 Å –4 ) 0.002 0.01 → 4.0 4.0 4.0 kdihedral (kcal mol –1 rad –2 ) 5 200 200 200 repel 0.9 0.9 → 0.75 0.75 0.80 a The temperature is maintained by coupling to a bath (16) with a coupling constant of 10 ps –1 . tion greater than approx 1 Å is an indication that there is a serious problem, especially if any such violation occurs repeatedly across the ensemble of structures. It is generally useful to identify the region of structures where these restraints are violated and critically inspect them using interactive computer graphics and check the corresponding NOE assignment and volume integration. 2. Atomic root-mean-square deviations (RMSD): The RMSD of the ensemble from the mean of the ensemble is a test that is commonly used to determine the “precision” of the structures, or how close the calculated structures are to each other. 3. Torsion angle distributions: Plots of the φ, ψ, χ i -torsion angles vs amino acid sequence of the protein and Ramachandran plots are useful to analyze local conformations and this allows assessment of the local geometry of the structures. 4. Quality assessment: Programs are available to perform a “quality check” of the structures derived from NMR data, for example, PROCHECK-NMR (17) and WHATIF (18). These software packages assess the quality of structures by comparing structural parameters with their values in databases derived from high resolution X-ray structures. 3.1.1.2. ADDITIONAL RESTRAINTS FOR STRUCTURE REFINEMENT Additional structural restraints can be added to the distance and torsion angle restraints for structure calculation in the final refinement of the structures. For example, 13 C secondary chemical shifts (19), which are related to backbone φ and ψ angles, 1 H chemical shifts (20,21), which are influenced for example by short-range ring-current effects from aromatic groups, magnetic anisotropy of C = O and C–N bonds (22) and residual dipolar couplings that give informa-
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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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Page 237 and 238: 218 Drakenberg sands, of Hertz broa
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Page 241 and 242: 222 Drakenberg Fig. 2. Measurement
Page 243 and 244: 224 Drakenberg Fig. 3. (A) 43 Ca NM
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Page 251 and 252: 232 Weljie and Heringa Table 1 Amin
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Page 265 and 266: 246 Weljie and Heringa much larger
Page 267 and 268: 248 Weljie and Heringa ment require
Page 269 and 270: 250 Weljie and Heringa 10. Kawasaki
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
Page 277 and 278: 258 Li et al. 7. Mineral Mixture (s
Page 279 and 280: 260 Li et al. 3. When the 45% D 2O/
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Page 293 and 294: 274 Mal et al. molecular dynamics a
Page 295 and 296: 276 Mal et al. Assuming a rigid bod
Page 297 and 298: 278 Mal et al. assign (segid A and
Page 299 and 300: 280 Mal et al. References 1. Drenth
Page 301 and 302: 282 Mal et al. 33. Jeener, J., Meie
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Page 305 and 306: 286 Werner et al. Our research has
Page 307 and 308: 288 Fig. 1. (A) T 1, T 2 and the he
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Page 317 and 318: 298 Werner et al. 9. Reinhardt, D.
Page 319 and 320: 300 Werner et al. 39. Press, W. H.,
Page 321 and 322: 302 Boyd et al. utilize residual di
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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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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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332 Kobayashi 0.1% TFA at a flow ra
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334 Kobayashi Both recombinant S-10
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336 Kobayashi Fig. 6. Affinity chro
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340 Walsh et al. verts CaM from an
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342 Walsh et al. 11. Bovine serum a
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344 Walsh et al. Fig. 1. CaM-depend
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348 Walsh et al. 3.5. NOS Reduction
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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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METHODS IN MOLECULAR BIOLOGY TM •
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