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Solution Scattering 155 structure. They further show that TnI undergoes a compaction upon addition of Ca 2+ . The measurements of the radius of gyration for TnI in the ternary troponin complex indicate a more compact structure than was observed for the binary complex, suggesting that of TnT influences the TnC–TnI interaction. The TnC–TnI interaction has proven very difficult to study, and there is a growing amount of apparently contradictory data using different experimental approaches. One difficulty the system has for study using scattering techniques is that there are no high-resolution structural data on TnI and its structure is very unusual. TnI is not a “typical” globular protein, and therefore cannot be modeled simply as an ellipsoid to aid in the interpretation of scattering data. Based on the neutron data for the binary complex, a model was developed (27) in which TnI forms a superhelical structure around the 4Ca 2+ /TnC extending into an incomplete donut shaped structures that project beyond the TnC at each end. The diameter of the TnI central spiral is 12 Å, close to that expected for an α helix, and it passes through or near the two hydrophobic clefts in each globular domain of 4Ca 2+ /TnC. Based on this model, it was proposed that the C-terminal domain of TnC anchors TnI while the N-terminal domain alternately binds and releases TnI in response to the Ca 2+ signal. Recent data from NMR, crosslinking FTIR, and crystallography have yielded a high-resolution model for Tn(–TnI) that is based on the earlier neutron model (28). Thus, although there remains much work to be done before we fully understand the complex interactions within the troponin complex, the neutron scattering experiments on TnC–TnI (25) and troponin (26) have yielded some important consensus conclusions. In the presence of the intact TnI, TnC has a fully extended structure that is very similar to its crystal structure. Further, TnI has an even more elongated structure, although the details of that structure show a dependence on TnT and Ca 2+ . 4. Notes 1. <strong>Protein</strong> aggregation and the importance of accurate protein concentration determinations and I0 calibration: Nonspecific protein aggregation can be fatal in a small-angle scattering experiment. Analysis of scattering data from solutions containing aggregates will give structural parameters that are systematically larger than the correct values. Because the scattering signal is proportional to the square of the molecular weight of the scattering particle, even small amounts of aggregated material in a solution will bias the data very severely toward the aggregated species. If it is severe enough, aggregation can be seen in the very small angle scattering data as a deviation from linearity in the Guinier plots (see Subheading 3.5.1.). However, samples with small amounts of aggregation can yield perfectly linear Guinier plots. Another signature of aggregation can be seen in the concentration dependence of the scattering data. For a homogeneous, monodisperse solution of protein molecules, the shape of the scattering profile should be either
156 Trewhella and Krueger concentration independent or show evidence of suppression of the lowest-Q data because of interparticle interference (see Subheading 3.3.). This suppression leads to increasingly underestimated structural parameters with increasing concentration. If the concentration dependence of the structural parameters goes in the opposite direction (i.e., increasing structural parameters with increasing concentration), then it is evidence for concentration-dependent aggregation, and extrapolation of the data to infinite dilution is unlikely to yield the true structural parameters of the monomeric protein. The best guard against being misled by aggregation is to place the scattering data on an absolute scale in order to obtain an accurate measure of the molecular weight of the scattering particle (29). Alternatively, the forward scattering from the protein of interest can be calibrated using a standard protein known to be monodisperse. A good protein standard is one that has a molecular weight close to the value of the protein you wish to study, and whose concentration can be accurately determined. A protein with a well-determined UV extinction coefficient can be a good choice. Apoferritin has been used as a standard, as has lysozyme (5). However, many proteins are not suitable for use as a standard at synchrotron intensities because of radiation induced aggregation and standards must be reevaluated for each new instrument. 2. Hydration layer effects: Macromolecules in aqueous solution have water molecules at their surfaces that can form a “hydration layer” with a mean scattering density that is different from that of the bulk solvent. In terms of the scattering experiment, the protein solution then becomes a three component system (protein, hydration layer, bulk solvent; 28). The effect of the hydration layer is to give a scattering pattern the yields structural parameters that are larger than for the protein by itself. The size of the hydration layer effect depends upon the charge on the protein surface and the ionic strength of the solution. For highly charged molecules, like DNA for example, the effects are significant. For close to neutral proteins in dilute buffered salt solutions (
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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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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
Page 155 and 156: 136 Hicks et al. 5. Hayes, D. B. (M
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
Page 167 and 168: 148 Trewhella and Krueger analytica
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Page 171 and 172: 152 Trewhella and Krueger P(r) func
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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
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Page 187 and 188: 168 Doherty-Kirby and Lajoie Fig. 1
Page 189 and 190: 170 Doherty-Kirby and Lajoie Fig. 2
Page 191 and 192: 172 Doherty-Kirby and Lajoie 5. Alt
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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
Page 199 and 200: 180 Shaw 3.3.3. Purification 1. Con
Page 201 and 202: 182 Shaw 10. Hodges, R. S., Semchuk
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
Page 209 and 210: 190 Brokx and Vogel it through an a
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Page 215 and 216: 196 Berliner Fig. 1. Aqueous X-band
Page 217 and 218: 198 Berliner in the Bruker instrume
Page 219 and 220: 200 Berliner Fig. 3. ESR spectra of
Page 221 and 222: 202 Berliner Fig. 5. Low-temperatur
Page 223 and 224: 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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