Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Solution Scattering 149 Fig. 1. Ribbon representation of the backbone structure of CaM in the crystal structure (top left, 16) and in its complex with the peptide MLCK-I derived from NMR data (top right, 19). The plots show the P(r) functions, each scaled to the square of the molecular weight, calculated from the crystal structure of 4Ca 2+ /CaM (solid line), and measured using solution scattering from CaM (dashed line), 4Ca 2+ /CaM (open circles), and 4Ca 2+ /CaM/MLCK-I (closed circles). within each of the individual globular lobes and the second dominated by vector lengths between the globular lobes. The experimental P(r) measured for 4Ca 2+ /CaM in solution shows a reduction in the number of longer vector lengths compared with that calculated for the crystal structure, and the second peak has
150 Trewhella and Krueger collapsed to a shoulder at a shorter average vector length. These changes indicate that the two lobes, on average, are closer together in solution and are reflected in the R g values that go from 22.8 Å for the crystal structure to 21.3 Å for the solution form. The apo-CaM P(r) is only slightly more compact than the Ca 2+ -saturated form, giving an R g value of 19.6 Å. The CaM/MLCK-I complex has a symmetric P(r) with a single peak and gives an R g value of 16.4 Å indicating a much more compact, globular shape. The d max value is also smaller, by about 20 Å, indicating that the globular lobes of CaM in the complex must come into close contact with each other. These scattering data were interpreted as indicating that CaM wrapped itself around the MLCK-I peptide by using the flexibility in the interconnecting helix region. Subsequent NMR experiments (19) confirmed the conformational collapse observed in the scattering involves the globular calcium-binding domains of CaM folding around the MLCK-I peptide that forms an amphipathic helix. The NMR data further show the complex is stabilized via hydrophobic interaction between the peptide and the hydrophobic clefts exposed upon Ca 2+ -binding to each globular domain of CaM. The scattering data have thus contributed to our view of the highly conserved, multifunctional CaM being designed for both specific binding to targets as well as diversity in the set of targets it must regulate. Many of the CaM target sequences are approx 20 residues in length and characteristically have a high propensity for forming an amphipathic helix, with large hydrophobic side chains spaced 12 residues apart (reviewed in ref. 16). There is, however, considerable sequence variability along the length of the peptide. The large hydrophobic side chains act as recognition and binding sites for the hydrophobic clefts, whereas the interconnecting helix region provides the flexibility to accommodate a variety of stereo-chemical surfaces. A number of CaM/peptide studies indicate that the conformational collapse observed in the CaM/MLCK-I example is a common feature of a number of Ca 2+ –CaM activation mechanisms (reviewed in refs. 16 and 18). However, there are no high-resolution structures of CaM complexed with any one of the more than 30 enzymes it is known to regulate, leaving a number of questions about the applicability of the results of the CaM/peptide studies to the enzyme activation mechanism. Would the extensive network of interactions between the CaM-binding and autoinhibitory sequences of MLCK inhibit the formation of the compact structure seen in the CaM/peptide complexes? Would the presence of other structural elements of the enzyme sterically hinder its formation? Small-angle neutron contrast variation studies answered these questions definitively and revealed the first structural view of CaM complexed with a catalytically active enzyme, MLCK (11,12). Structural models for the MLCK/deuterated-CaM complex with and without bound substrates were obtained (see Fig. 2). 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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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 133 and 134: 114 Lopez and Makhatadze Fig. 1. Th
Page 135 and 136: 116 Lopez and Makhatadze 6. Buffers
Page 137 and 138: 118 Lopez and Makhatadze The excess
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
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
Page 159 and 160: 140 Trewhella and Krueger of the sc
Page 161 and 162: 142 Trewhella and Krueger Table 1 C
Page 163 and 164: 144 Trewhella and Krueger beam, or
Page 165 and 166: 146 Trewhella and Krueger At very l
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Page 181 and 182: 162 Doherty-Kirby and Lajoie metal
Page 183 and 184: 164 Doherty-Kirby and Lajoie two Ca
Page 185 and 186: 166 Doherty-Kirby and Lajoie 7. Pep
Page 187 and 188: 168 Doherty-Kirby and Lajoie Fig. 1
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Page 195 and 196: 176 Shaw Fig. 1. Ribbon drawing of
Page 197 and 198: 178 Shaw 2.3. Other 1. Chemicals fo
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Page 203 and 204: 184 Brokx and Vogel Table 1 An Over
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Page 207 and 208: 188 Brokx and Vogel Fig. 2. 20% SDS
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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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