Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Cadmium-113 and Lead-207 NMR Studies 205 17 Cadmium-113 and Lead-207 NMR Spectroscopic Studies of Calcium-Binding Proteins Teresa E. Clarke and Hans J. Vogel 1. Introduction Ideally, direct NMR spectroscopic studies of the structure and dynamics of a metal binding site would utilize the ion that is naturally bound to the protein. For calcium-binding proteins, the NMR active isotope 43 Ca is a quadrupolar (I = 7/2) nucleus that produces broad peaks when bound to a protein (1–4). Although much useful information can be gleaned from studies of this nucleus (3,4), it is impossible to resolve the signals for individual calcium-binding sites in a protein with multiple-binding sites. However, isomorphous replacement of the calcium ion with an ion with more favorable NMR properties often allows the resolution of NMR signals for individual sites and provides valuable insight into the coordination of the ion within the binding site from the observed shifts. 113 Cd, and to a lesser extent, 207 Pb, are two such I = 1/2 metal nuclei that have been successfully used to characterize the Ca 2+ -binding properties of a variety of metalloproteins (5–8). Although Cd 2+ and Pb 2+ are toxic metal ions and not of direct biological relevance, they can effectively substitute for Ca 2+ in metalloproteins, allowing them to retain similar structures and function. Cd 2+ has a filled d-shell orbital and can form complexes with a variety of conformations and number of ligands. Relative to Ca 2+ (r = 0.99 Å), Cd 2+ has a very similar ionic radius (r = 0.97 Å), whereas Pb 2+ is slightly larger (r = 1.20 Å). For both 113 Cd and 207 Pb, the chemical shift of the NMR signal is highly dependent on the ligand environment of the metal ion. An increase in the polarizability of the ligand results in a decrease in nuclear shielding (i.e., S < N < O), such that oxygen-coordinated metal signals resonate the furthest upfield. Typi- 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 205
206 Clarke and Vogel cal calcium-binding sites in proteins are usually made up of oxygen ligands, so their NMR signals are found in the upfield portion of the spectra. The large chemical shift window available in both 113 Cd and 207 Pb NMR can also distinguish subtle differences between motifs in Ca 2+ -binding sites (3–8). This chapter will focus on simple one-dimensional (1D) 113 Cd and 207 Pb NMR spectroscopic techniques in order to demonstrate the ease of distinguishing between different calcium-binding motifs. Other NMR studies involving these nuclei, including solid-state NMR studies or 2D heteronuclear NMR spectroscopy, can in principle, also be useful to characterize Ca 2+ -binding proteins, but are beyond the scope of this work. Cadmium has been substituted for a wide range of metal ions other than Ca 2+ , and the reader is referred to a number of comprehensive reviews on the use of cadmium-113 NMR spectroscopy for studies of such metalloproteins (5–7). 2. Materials 1. Acid-washed glassware and plasticware should be prepared by washing extensively in 1 M HCl and rinsing thoroughly in distilled H2O. 2. For calcium removal from the protein, one can use a 10 DG column (Bio-Rad Laboratories) and 50 mM ammonium bicarbonate and 0.5 M ethylenediaminetetracetic acid (EDTA), pH 8.0. Alternately, the protein can be passed through a Chelex column, eluting with 50 mM ammonium bicarbonate (see Note 1). 3. A widebore Bruker AM400 instrument equipped with a 10-mm broadband probe with variable temperature control capabilities (see Note 2). Higher or lower field instruments can be used with appropriate changes to the parameters (see Note 3). 4. A 100-mM stock solution of 113Cd(ClO4) 2 is prepared by dissolving the 113CdO (95%) (Cambridge Isotope Laboratories) in concentrated HCl, heating to dryness under a stream of nitrogen with low heat, and dissolving the residue in the appropriate volume of 200 mM HClO4 in D2O (99.9%) (Cambridge Isotope Laboratories) (9). Alternatively, a 100-mM solution of 113CdSO4 can be prepared as the stock solution (see Note 4) by dissolving in 3 M H2SO4, heating to dryness as above, and taking the powder up in a correct volume of 50/50 D2O/H2O and neutralizing with NaOH to pH 6.0 (10). 5. A 100-mM stock solution of 207Pb(NO3) 2 (91.6%) (Oak Ridge National Laboratories) is made up in D2O to be used as a titrant and chemical shift reference (8). 6. Materials for limited proteolysis of the Ca2+ -binding proteins, as described in Chapter 15 in this volume (11). 3. Methods 3.1. Sample Preparation The appropriate amount of a purified, lyophilized calcium-binding protein is dissolved in 1 mL 50 mM ammonium bicarbonate and 150 µL 0.5 M EDTA, pH 8.0, and desalted by two passes through a 10 DG column, then freeze dried.
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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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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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314 Boyd et al. 4. Downing, A. K.,
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316 Boyd et al. 35. Schulte-Herbrug
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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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