Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Steady-State Fluorescence Spectroscopy 79 Fig. 4. Potassium iodide Stern-Volmer quenching plots of a synthetic peptide encompassing the CaM-binding domain of skeletal myosin light-chain kinase alone (filled rectangles), and in complexes with wild-type calmodulin (filled triangles), a mutant calmodulin where the C-terminal methionine residues were replaced with leucine (filled ovals), and a CaM where the methionine residues were replaced by selenomethionine. Notice that the free peptide shows a much greater degree of solvent exposure than the CaM-target complexes as evidenced by the increased slope (10,12). The slope in a Stern-Volmer quenching plots provides the Stern-Volmer quenching constant K SV. 2. Materials 1. Wavelength-scanning spectrofluorimeter capable of excitation at 295 nm (see Note 1) and detection of fluorescence emission between 300 and 450 nm, such as the Hitachi F-2000. Ideally the instrument should either be connected to a microcomputer or have data transfer capability to facilitate subsequent analysis of the data. 2. Quartz cells that have two transparent faces; one for excitation and one for emission. Generally these faces will be at 90° to each other and cells are available as rectangular cuvets with four clear sides. For cells with a path length of 1.0 cm, the sample volume needed is approx 3.0 mL. 3. Standard buffers chosen to meet pH and ionic requirements of the protein(s) under study, bearing in mind that absorption of buffer components in the UV range is undesirable. The absorption of the buffer can be measured using a standard UV/
80 Weljie and Vogel VIS spectrophotometer and acquiring an absorption spectrum between 250 and 320 nm. It may also be worthwhile to acquire a UV/VIS spectrum of the buffer between 300 and 450 nm to ensure that there is no transfer of tryptophan-fluorescence emission to the buffer. If one is examining the interaction of charged species, the addition of 50–100 mM KCl or NaCl may be beneficial to prevent nonspecific interactions. Finally, phosphate buffers are to be avoided in studies involving Ca 2+ because of the precipitation of calcium phosphate solid. 4. For quenching studies, a saturated stock solution of the chosen quenching agent is required. For example, a stock solution of potassium iodide can be made by adding 30.0 g of KI to 21.0 mL of water, to form 30.0 mL of a 6.0 M solution (see Note 2). 3. Method 3.1. Sample Preparation 1. Purified, dry protein is weighed and then dissolved in the chosen buffer. Care must be taken to ensure that the pH does not vary significantly. This is especially necessary for calcium-binding proteins which often have significant numbers of acidic residues in the calcium binding regions (see Note 3). 2. The concentration of the sample must be quantitatively determined, as the dry mass is not a reliable indicator of protein mass. The method of choice for such concentration determinations of proteins (or peptides) without tryptophan and tyrosine is quantitative amino acid analysis. If the protein sequence is known, the absorbance at 280 nm can be used if the protein contains tryptophan or tyrosine residues. The extinction coefficient can be calculated as: ε280 = x εtyr + y εtrp + z εcys where x, y, and z are the numbers of each residue in the sequence, and the value of εtyr is 1280 M –1cm –1 , εtrp is 5690 M –1cm –1 , and εcys (cystine) is 120 M –1cm –1 . (13) (see Note 4). 3. If a number of samples are to be made of the same protein or peptide, a 100X stock solution can be made, and then a sample pool made with a volume slightly greater than the total volume of all samples. For example, if 10 3.0 mL samples of protein are needed, then a 30.5-mL pool might be made, and each sample made from this pool. This will help ensure that the concentration of each sample remains consistent, which is especially important when comparing spectra of the same species under different conditions. Ensure that the volume used also takes into account other reagents that might be added, such as calcium stock solutions (see Note 5 for suggested stock concentrations). 4. Samples involving binding (calcium and or protein/peptide ligands) should be allowed to come to a complete equilibrium prior to spectrum acquisition. For stable species, samples can be prepared the day prior to use, and then stored at 4° overnight. Ensure that the samples are allowed to equilibrate to room tempera-
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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
Page 47 and 48: 28 Haiech and Kilhoffer where γ is
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Page 51 and 52: 32 Haiech and Kilhoffer tein with i
Page 53 and 54: 34 Haiech and Kilhoffer Calmodulin
Page 55 and 56: 36 Haiech and Kilhoffer Fig. 3. Mec
Page 57 and 58: 38 Haiech and Kilhoffer We may sugg
Page 59 and 60: 40 Haiech and Kilhoffer 29. Stewart
Page 61 and 62: 42 Haiech and Kilhoffer 62. Becking
Page 63 and 64: 44 Martin and Bayley Fig. 1. Absorp
Page 65 and 66: 46 Martin and Bayley evaporation. C
Page 67 and 68: 48 Martin and Bayley 4. Set the tem
Page 69 and 70: 50 Martin and Bayley Fig. 2. Near-U
Page 71 and 72: 52 Martin and Bayley discussed by M
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 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 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 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
Page 123 and 124: 104 Julenius Fig. 1. Under conditio
Page 125 and 126: 106 Julenius 3. Mix 50 µL of NHS s
Page 127 and 128: 108 Julenius Fig. 3. A typical sens
Page 129 and 130: 110 Julenius (2), is used in the Ia
Page 133 and 134: 114 Lopez and Makhatadze Fig. 1. Th
Page 135 and 136: 116 Lopez and Makhatadze 6. Buffers
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Page 141 and 142: 122 Lopez and Makhatadze Fig. 1. Is
Page 143 and 144: 124 Lopez and Makhatadze 2.5 mL are
Page 145 and 146: 126 Lopez and Makhatadze pendence o
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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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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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