Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Differential Scanning Calorimetry 115 Fig. 2. The partial molar-heat capacity of chicken ovomucoid at pH 3.3 obtained experimentally (thick solid line) deconvoluted into three independent transitions (thin solid lines) with the thermodynamic parameters for individual transitions shown. customary to use lower heating rates (30–60°/h). Fibrillar proteins such as collagen or myosin, which exhibit very narrow transitions, a heating rate of 10–20°/h is most suitable. 2. Materials 1. The DSC instrument designed to study biological systems must be extremely sensitive and require small amounts of the material, 0.1–1 mg/mL of protein solution. Currently there are two commercial DSC instruments, Nano-DSC from Calorimetric Science Corporation (Provo, UT) and VP-DSC from Microcal Inc. (Northhampton, MA). Both of these instruments are fully automated for control, data collection, handling, and analysis using PC computers. VP-DSC appears to have higher sensitivity and better baseline stability and is supplied with the superb ORIGIN graphics software. 2. Syringe with precut needle used to wash the cell and load the sample (provided by the manufacturer). 3. Spectrophotometer and quartz cells of different path length, for those cases in which the protein concentration is determined spectrophotometrically. 4. Dialysis bags with molecular weight cutoff depending on the molecular weights of the protein to be dialyzed. 5. Highly pure protein sample.
116 Lopez and Makhatadze 6. Buffers should be chosen for their low ionization enthalpy (and thus low-temperature dependence of pK a) such as glycine (pH 2.0–3.5), sodium acetate (pH 3.5–5.0), sodium cacodylate (pH 5.5–7.0), sodium phosphate (pH 6.0–7.5). 3. Methods 3.1. Instrument Preparation 1. The instrument should be turned on at least 12 h prior to the experiment and “thermal history” established by running the baseline scans with the cells filled with the buffer. 2. Calibration of the instrument should be done periodically (once a year) using the procedure provided by the manufacturer (see Note 1). 3.2. Sample Preparation 1. Purified protein should be extensively dialyzed (with several changes of buffer every 6 h or more) against corresponding buffer. 2. Prior to the experiment insoluble particle and dust should be eliminated by centrifugation at 13,000g. Filtration of the protein solution is not recommended. 3. Measure protein concentration (see Note 2). 3.3. Data Collection 1. Thoroughly wash both cells with buffer from the last dialysis and fill them with the buffer. It is important to avoid any air bubbles trapped in the cell. For this, after filling up the syringe, pump out all air bubbles. Insert the needle into the calorimetric cell (needles are precut to a specific length so that the tip of the needle is barely above the bottom of the cell) and slowly lower the plunger until the solution appears in the overflow reservoir. At this point, start abruptly pumping solution in and out of the cell. This abrupt pumping will force trapped air bubbles out from the cell. 2. Fill both cells with the buffer and run buffer/buffer scan. 3. Refill the sample cell with the protein solution and run protein/buffer scan. 4. Rescan to check the reversibility of the unfolding (see Note 3). 3.4. Data Analysis app 1. Subtract the protein/buffer scan from the buffer/buffer scan to obtain ∆Cp (T), the heat-capacity difference between sample and reference cells at temperature T. app 2. Convert ∆Cp (T) into the partial heat capacity of the protein at temperature T, exp Cp, pr (T) as: exp Cp, pr (T) = Cp,H2O / V — H2O · V — app pr – ∆Cp (T) / mpr where V — pr is the partial volume of the protein, mpr is the mass of the protein in the calorimetric cell, V — H2O (T) is partial molar volume of aqueous buffer, and Cp,H2O is the heat capacity of aqueous buffer. The partial volume of the protein, V — pr can be calculated from the amino acid composition of the protein using an
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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
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
Page 97 and 98: 78 Weljie and Vogel Fig. 3. Steady-
Page 99 and 100: 80 Weljie and Vogel VIS spectrophot
Page 101 and 102: 82 Weljie and Vogel 4. A series of
Page 103 and 104: 84 Table 1 Advanced Fluorescence Me
Page 105 and 106: 86 Weljie and Vogel References 1. L
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Page 109 and 110: 90 Johnson and Tikunova is removed
Page 111 and 112: 92 Johnson and Tikunova function of
Page 113 and 114: 94 Johnson and Tikunova Fig. 2. Rat
Page 115 and 116: 96 Johnson and Tikunova Fig. 3. Rat
Page 117 and 118: 98 Johnson and Tikunova Fig. 4. Ca
Page 119 and 120: 100 Johnson and Tikunova cence inte
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
Page 127 and 128: 108 Julenius Fig. 3. A typical sens
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Page 137 and 138: 118 Lopez and Makhatadze The excess
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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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Page 165 and 166: 146 Trewhella and Krueger At very l
Page 167 and 168: 148 Trewhella and Krueger analytica
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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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