Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Spatial Distribution of Ca 2+ -<strong>Binding</strong> <strong>Protein</strong>s 391 Fig. 4. Amino acid sequence of human calmodulin showing peptide fragments expected from a typical tryptic digest, which catalyzes the hydrolysis of lysyl and arginyl peptide bonds. Lys 75 and Lys 148 are shown in bold to show that these were the two main 5-DTAF labeling sites in doubly labeled FL-calmodulin and Lys 75 only in singly labeled FL-calmodulin. Fig. 5. Electrospray mass spectrum of the desalted, freeze dried peptide from peak 7 isolated by HPLC dissolved in 10% acetonitrile 0.1% formic acid solvent. The first mass (1843.70 (± 0.37) Da) relates to peptide contamination from the preceeding peak, whereas the second mass (1901.91 (± 0.41) Da) relates to peptide fragment 75–86 with the 5-DTAF probe intramolecularly cyclized between two amino groups by substitution of both chlorines.
392 Török et al. Table 1 Peptide Fragments Isolated by HPLC on a Vydac Semipreparative Column; Peptides were Analyzed by Electrospray and Nanospray Mass Spectrometry Observed Modified Expected Peak Average Mass Sequence Residues Average Mass 1 1092.5 T8 1093.1 (77–86) 2 521.5 T9 521.6 (87–90) 3 803.9 T4 804.9 (31–37) 4 1753.7 T10–11 1753.9 (91–106) 5 1562.8 T1 Acetyl Group 1562.8 (1–13) on Alanine 1 6 1843.9 T2–T3 1843.9 (14–30) 7 1901.9 75–86 *5-DTAF 1902 cyclized 8 2400.5 T12 (CH 3) 3 on 2402 (107–126) Lysine 115 9 864.1 75–77 5-DTAF on 863.6 Lysine 75 10 2489.1 T13 2490 (127–148) 11 2302.5 T2–T3 5-DTAF 2301.9 (14–30) on Lysine 21 12 a 2951 T13 5-DTAF 2948.7 (127–148) on Lysine 148 13 4070.7 T5 4071.5 (37–74) a Doubly labeled only intramolecularly cyclized between two amino groups by substitution of both chlorines (see Fig. 6). Analysis of peak 9 by nanospray mass spectrometry shown in Fig. 7A, shows a monoisotopic mass of 864.1, which identifies peptide fragment 75–77 with a 5-DTAF label on one of the two lysines. In Fig. 7B, we can see the entire amino acid sequence and which lysine residue is labeled with 5-DTAF. In this case, the label is on Lys 75 and not on Lys 77 because the b ions (fragmentation occurring at the amide backbone to produce acylium ions) and the y” ions (C-terminal ions involving hydrogen rearrangement) would produce different masses as outlined in Fig. 8 (see Note 2). The reaction mechanism that gives these two different peptides is shown in Fig. 6. As aforementioned, in the
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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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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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Page 359 and 360: 340 Walsh et al. verts CaM from an
Page 361 and 362: 342 Walsh et al. 11. Bovine serum a
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Page 379 and 380: 360 Hughes et al. range, repeat ste
Page 381 and 382: 362 Hughes et al. Table 1 Examples
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Page 385 and 386: 366 Persechini of the different CaM
Page 387 and 388: 368 Persechini Fig. 1. Schematic re
Page 389 and 390: 370 Persechini 2. 1 L of Terrific b
Page 391 and 392: 372 Persechini Fig. 4. Titration wi
Page 393 and 394: 374 Persechini Table 1 Parameters f
Page 395 and 396: 376 Persechini Fig. 6. The [Ca 2+ -
Page 397 and 398: 378 Persechini determined if the di
Page 399 and 400: 380 Persechini relatively insensiti
Page 401 and 402: 382 Persechini 18. Chafouleas, J. G
Page 403 and 404: 384 Török et al. actions in the c
Page 405 and 406: 386 Török et al. 3. The reaction
Page 407 and 408: 388 Török et al. Fig. 2. Electros
Page 409: 390 Török et al. Fig. 3. Peptides
Page 413 and 414: 394 Török et al. Fig. 7. Nanospra
Page 415 and 416: 396 Török et al. Fig. 9. Nanospra
Page 417 and 418: 398 Török et al. Fig. 11. Electro
Page 419 and 420: 400 Török et al. 3 µM FL-calmodu
Page 421 and 422: 402 Török et al. Fig. 14. Localiz
Page 423 and 424: 404 Török et al. Fig. 16. Indicat
Page 425 and 426: 406 Török et al. the significantl
Page 427 and 428: 408 Török et al.
Page 429 and 430: 410 Index substitutes, see Fluoresc
Page 431 and 432: 412 Index Stern-Volmer plot, 78, 81
Page 433 and 434: 414 Index Phenothiazines, see Calmo
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