Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Fluorescence Methods for Ca 2+ Exchange 97 tion can be minimized by using more concentrated Ca 2+ stocks. If necessary, the fluorescence intensity can be corrected for dilution at each point in the titration by multiplying the observed fluorescence intensity after the addition by the dilution factor (the ratio of the volume of the solution after the addition to the volume of the solution before the addition). 3. Always run spectra — Even though it takes a few additional minutes, it is essential to run an emission (or excitation spectra) of your solution both before and after the titration. This assures that the fluorescence change you are following results from a change in the fluorescence of the fluorophore and is not a result of changes in light scattering. These spectra allow one to determine the wavelength at which the largest fluorescence change occurs and this can be used to increase signal to noise in the titration. Although it is generally best to do the titration using the emission wavelength where the greatest fluorescence change occurs, in more turbid solutions, scattering can be a problem. When this is the case, the contribution of scatter to emission can be reduced by selecting an emission wavelength that is further removed from the excitation wavelength and where the fluorophore still undergoes a large fluorescence change with [Ca 2+ ]. 4. Calibration of CaCl 2 stocks and EGTA buffers — The most critical points for accurate Ca 2+ titrations is the precise control of free [Ca 2+ ] using Ca 2+ chelators like EGTA or ethylenediaminetetracetic acid (EDTA). It is important to use sufficient Ca 2+ buffer (EGTA or EDTA) so that the small amount of endogenous Ca 2+ bound to the protein (generally 1 µM or less) being titrated does not affect free [Ca 2+ ]. We typically conduct titrations in the presence of 2 mM EGTA and use 200 mM MOPS (or HEPES) at pH 7.0 to prevent decreases in pH as Ca 2+ binds and releases protons from EGTA. Other buffers can be used as long as they do not contribute or bind Ca 2+ . Any desired ionic strength can be used as long as its affect on EGTAs affinity for Ca 2+ are considered by the pCa computer program. Several computer programs are available for determining the free Ca 2+ , as a function of total added Ca 2+ , for a given [EGTA] or [EDTA] at various temperatures, ionic strengths and pH (see Notes 9 and 10). Obviously, the stock [CaCl 2] and [EGTA] must be accurately made and this is facilitated by heating the powders of both to 80–90°C for 10 min to remove water before weighing. The CaCl 2 and EGTA stocks and the ability to accurately vary free Ca 2+ as a function of added Ca 2+ should always be tested and calibrated by conducting Ca 2+ titrations of a fluorescent Ca 2+ indicator like Quin-2, Fura-2, or Fluo-3. Because each of these Ca 2+ indicators bind one mole of Ca 2+ per mole of indicator with known K ds, if the fit of the Ca 2+ dependent change in fluorescence gives the appropriate K d and Hill coefficient (1.0) you can be confident that you are accurately controlling free Ca 2+ . Figure 4 shows typical Ca 2+ dependent increases in Quin-2, Fura-2, and Fluo-3 fluorescence. Half-maximal increases occurred at pCa 7.21 (62 nM) for Quin-2, pCa 6.86 (138 nM) for Fura-2, and pCa 6.36 (430 nM) for Fluo-3. All of the indicators exhibited Hill coefficients of 1.03 ± 0.04. These K ds are essentially identical to the K ds for Quin-2 (60 nM), Fura-2 (145 nM) and Fluo-3 (390 nM), reported by their supplier in the Molecular Probes
98 Johnson and Tikunova Fig. 4. Ca 2+ titrations of Quin-2, Fura-2, and Fluo-3. Increasing concentrations of Ca 2+ were added to 1 µM of each indicator in 1 mL of buffer (200 mM MOPS, 90 mM KCl, 2 mM EGTA) at 22°C. Excitation was at 330, 340, and 490 nm and emission was at 495, 510 and 525 nm for Quin-2, Fura-2, and Fluo-3, respectively. 100% fluorescence increase corresponds to 7.8-, 2.6-, and 52-fold increases for Quin-2, Fura-2, or Fluo-3, respectively. Each data point represents an average of 3 titrations ± S. E. handbook (12). Thus, Ca 2+ titrations of any of these indicators provides an easy means of verifying the accuracy of your CaCl 2 and EGTA stocks. Errors in either K d or Hill coefficient can indicate inappropriate control of pCa. If this occurs, then the concentrations of the EGTA or CaCl 2 stocks must be off and should be adjusted till accurate Ca 2+ dependent increases in indicator fluorescence are obtained. The concentration of the CaCl 2 stock (generally 0.5 M) can also be verified by atomic absorption spectroscopy. If the CaCl 2 stock is correct, errors in the [EGTA] are probably responsible for deviant Ca 2+ titration. After your CaCl 2 and EGTA buffers have been made and calibrated, they can be broken into aliquots and stored frozen, in plastic, for years. 4.2. Fluorescence Methods to Measure Ca 2+ Dissociation Rates from <strong>Protein</strong>s 5. For very rapid kinetic reactions, some of the fluorescence change may be lost during in the mixing time of the instrument. For example in Fig. 2, F19W + Ca 2+ had a fluorescence intensity of 4.6 V (control shot) yet the observable decrease in
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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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Page 67 and 68: 48 Martin and Bayley 4. Set the tem
Page 69 and 70: 50 Martin and Bayley Fig. 2. Near-U
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Page 73 and 74: 54 Martin and Bayley References 1.
Page 75 and 76: 20 Dean, Kelsey, and Reik
Page 77 and 78: 58 Fabian and Vogel are equipped wi
Page 79 and 80: 60 Fabian and Vogel The penetration
Page 81 and 82: 62 Fabian and Vogel perature, ionic
Page 83 and 84: 64 Fabian and Vogel Fig. 3. IR spec
Page 85 and 86: 66 Fabian and Vogel Fig. 4. Lower t
Page 87 and 88: 68 Fabian and Vogel These correlati
Page 89 and 90: 70 Fabian and Vogel Fig. 5. Amide I
Page 91 and 92: 72 Fabian and Vogel 4. ATR-FTIR spe
Page 93 and 94: 74 Fabian and Vogel 25. Georg, H.,
Page 95 and 96: 76 Weljie and Vogel Fig. 1. Simplif
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Page 99 and 100: 80 Weljie and Vogel VIS spectrophot
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Page 103 and 104: 84 Table 1 Advanced Fluorescence Me
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Page 111 and 112: 92 Johnson and Tikunova function of
Page 113 and 114: 94 Johnson and Tikunova Fig. 2. Rat
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Page 121 and 122: 102 Johnson and Tikunova 6. Johnson
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Page 125 and 126: 106 Julenius 3. Mix 50 µL of NHS s
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Page 143 and 144: 124 Lopez and Makhatadze 2.5 mL are
Page 145 and 146: 126 Lopez and Makhatadze pendence o
Page 147 and 148: 128 Hicks et al. equipped with a pu
Page 149 and 150: 130 Hicks et al. Fig. 2. Debye plot
Page 151 and 152: 132 Hicks et al. and 1.0 mg/mL for
Page 153 and 154: 134 Hicks et al. Fig. 3. Sedimentat
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Page 157 and 158: 138 Trewhella and Krueger This chap
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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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20 Dean, Kelsey, and Reik
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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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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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METHODS IN MOLECULAR BIOLOGY TM •
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