Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Deconvolution of <strong>Calcium</strong>-<strong>Binding</strong> Curves 35 of the parameters, experimental data obtained, either by measuring the number of ligands bound to the protein or by following a spectroscopic signal as a function of the ligand concentration, can be fitted to the model. One of the main assumption of the model of Wang is the independence of the two lobes. If this assumption is true, the behavior of whole calmodulin must be the sum of the behaviors of its individual lobes (which can be obtained by proteolytic digestion [51,57]). Some studies concluded that there was little or no interaction between the N-terminal and C-terminal halves of calmodulin. This was based on comparisons of titration curves of whole calmodulin vs those of isolated domains (58–60). Other studies, using mutated calmodulins, were in favor of an interaction between the two domains (61–64). A controversy arose on this issue, as one cannot be sure that the tryptic fragments generated retain their original structure, nor that mutagenesis does not induce minor changes in the protein structure. It is only recently that work performed on whole calmodulin set up his controversy and clearly established that the two lobes of calmodulin were not independent (65–68). Also, in the presence of target proteins or peptides, the cooperativity between the two lobes of CaM markedly increases. To get more insight into the mechanism of Ca 2+ -binding to calmodulin, we decided in 1986 to build isofunctional mutants with an internal reporter group (64,69,70) that will give us access to the occupancy of one specific site of the protein. Our strategy was based on the reasoning developed in Subheading 3. (see Fig. 2). As standard calmodulin does not harbor tryptophan residues in its structure, we introduced one tryptophanyl residue at specific positions in calmodulin (64). Results allowed us to confirm the model we proposed in 1981. This model is presented in Fig. 3. Each site exists in two different conformations: a conformation of high affinity for Ca 2+ (HC conformation) and a low-affinity Ca 2+ -binding conformation (LC conformation). In the apoform, only site III is in the HC conformation. Upon Ca 2+ occupancy of this site, site IV undergoes a conformational change toward a HC conformation, and so on, step by step, for the remaining sites. This model is basically described by four constants, the individual binding constants of each individual site in the HC conformation. If g1, g2, g3, and g4 are these constants, the equation describing Ca 2+ -binding to calmodulin can be written g 1 * (L) + 2 * g 1 * g 2 * (L) 2 + 3 * g 1 * g 2 * g 3 * (L) 3 + 4 * g 1 * g 2 * g 3 * g 4 (L) 4 v = —————————————————————————————— (18) 1 + g 1 * (L) + g 1 * g 2 * (L) 2 + g 1 * g 2 * g 3 * (L) 3 + g 1 * g 2 * g 3 * g 4 (L) 4 These constants (g i) correspond to the macroscopic constants of the Adair-Klotz Eq. 2. In this model, we have a direct interpretation of the macroscopic constants in terms of individual constants and coupling factors.
36 Haiech and Kilhoffer Fig. 3. Mechanism of Ca 2+ -binding to calmodulin. The big circles correspond to Ca 2+ , the thether to the helices and connecting peptides. At the start, all sites but site III, are in a conformation of low affinity for Ca 2+ (LC conformation). After each Ca 2+ - binding step, one specific site acquires the high-Ca 2+ -affinity conformation (HC conformation). Numbers correspond to the Ca 2+ -affinity constants.
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Methods in Molecular BiologyTM Biol
Page 3 and 4: M E T H O D S I N M O L E C U L A R
Page 5 and 6: © 2002 Humana Press Inc. 999 River
Page 8 and 9: Preface Calcium plays an important
Page 10 and 11: Contents Dedication ...............
Page 12: Contents xi 26 Enzymatic Assays to
Page 15 and 16: xiv Contents of Companion Volume 15
Page 17 and 18: xvi Contributors LESLIE D. HICKS
Page 19 and 20: 20 Dean, Kelsey, and Re
Page 21 and 22: 2 Yazawa
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Page 27 and 28: 8 Yazawa DuPont-NEN and the molar c
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Page 33 and 34: 14 Yazawa 12. Starovasnik, M. A., D
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Page 37 and 38: 18 Linse 7. 0.1 M EDTA. Dissolve 37
Page 39 and 40: 20 Linse iterate the other paramete
Page 41 and 42: 22 Linse tive to small alterations
Page 43 and 44: 24 Linse References 1. Tsien, R. Y.
Page 45 and 46: 26 Haiech and Kilhoffer to use the
Page 47 and 48: 28 Haiech and Kilhoffer where γ is
Page 49 and 50: 30 Haiech and Kilhoffer reporter gr
Page 51 and 52: 32 Haiech and Kilhoffer tein with i
Page 53: 34 Haiech and Kilhoffer Calmodulin
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
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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
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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
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86 Weljie and Vogel References 1. L
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20 Dean, Kelsey, and Reik
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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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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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