Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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<strong>Calcium</strong>-43 NMR 223 it is possible to find a calcium chelator with a binding constant for calcium similar to that of the protein, it is possible to determine the binding constant in a competition experiment (6). By using an equimolar concentration of chelator and protein Ca 2+ will be evenly distributed between chelator and protein when they have the same affinity for Ca 2+ . In ref. 6, the chelator affinity was finetuned to that of the protein (lysozymes and α-lactalbumin) by changing pH, Fig. 3. Using ethylenediaminetetracetic acid (EDTA) as chelator, it was in this way possible to determine Ca 2+ -binding constants from 1 × 10 6 to 2 × 10 7 M –1 . These values are two to three orders of magnitude above what can be reached by the direct titration method, but are in the same range as those determined by using fluorescent chelators (see Chapter 2, this volume). When the dissociation constant for calcium binding to a protein is close to the concentration used in the experiments, it can be determined from a titration experiment. However, if the calcium off-rate is not fast, compared to the relaxation rate of the bound ion, care has to be exercised to take this into account (25). For phospholipase A 2 (PLA 2) and prophospholipase A 2 (PPLA 2) the effect on exchange was taken into account by simultaneously fitting the data from a temperature dependence experiment and a concentration dependence experiment (17) (Fig. 4). This will result in the determination of both binding constant and kinetics of the calcium ion. Preferably the experiment should be executed on a single sample in the following way. (1) Start with as low a calcium concentration as possible for obtaining a reasonable spectrum in a reasonable time and titrate until the linewidth has been reduced to approximately half of the initial value. At this point, the protein should preferably be saturated with calcium to such a degree that a temperature-dependence in the binding constant has only a minor effect. (2) Perform the temperature dependence experiment on the same sample. (3) Repeat the last experiment in the titration experiment; and (4) complete the titration. If the protein is not thermostable, as will be seen from the nonidentity of the repeated experiment, separate samples for the titration and temperature dependence have to be used. When all exchange rates are fast on the NMR time scale, it is more straightforward to determine the binding constant directly from a concentration dependence in the linewidth. The temperature dependence will not result in any useful information in this case. As shown in refs. 6 and 17, 43 Ca NMR can also be used to determine pK a values of residues involved in the binding site. For the case of strong binding and slow exchange, the pK a-value can be calculated from the change in the intensity of the NMR signal from bound ions as a function of pH (6). For the case of weaker binding and fast exchange, the pH dependence in the line width can be used to calculate the pK a-value (17). Without taking the effect of the calcium binding into account, this treatment will of course result in apparent pK a-values.
224 Drakenberg Fig. 3. (A) 43 Ca NMR spectra of (A) bovine a-lactalbumin at pH 7.0; (B) human α-lactalbumin at pH 7.0; (C) equine lysozyme at pH 6.0; (D) pigeon lysozyme at pH 6.8. (B) Elucidation of dissociation constants (K D) for the above for proteins using the pH dependence of the affinity of EDTA for Ca 2+ . ( .... ) Equine lysozyme, K D = 1 × 10 6 M; (– –) pigeon lysozyme, K D = 1 × 10 –7 M; (– . . –) bovine and human α-lactalbumin, K D = 5 × 10 –8 M. Reproduced with permission from ref. 6. However, when the calcium affinity is known the effect from the calcium binding can be taken into account to obtain a “true” pK a-value by using Eq. 6. ∆ν 1/2 obsd = ∆ν1/2*K[E]/(1 + K[E]) (6)
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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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Page 275 and 276: 256 Li et al. In order for these ex
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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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