Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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FTIR Spectroscopy of Calcium-Binding Proteins 71 3.5. Time-Resolved FTIR Spectroscopy Although FTIR spectroscopy has certain limitations in the quantification of protein secondary structure, it represents an excellent tool for monitoring, in relative terms, changes in the conformation of proteins under equilibrium (20) and nonequilibrium conditions (21). The highest sensitivity is provided by techniques that can induce the conformational change with minimal sample manipulation and without removing it from the instrument; the most powerful approach involves the use of light to trigger the event. Originally, these techniques were developed for the investigation of light-induced reactions in photoreactive proteins. They can be very specific down to the level of single functional groups in a large molecule with nanosecond time resolution applying sophisticated experimental setups (22,23). The use of caged compounds creates unique possibilities to initiate reactions by light (24). For studies of calcium-binding proteins, photolabile derivatives of cation chelating reagents such as DM-nitrophen are of particular interest. DM-nitrophen consist of a photolabile group linked to an EDTA molecule. The EDTA moiety of the molecule is split into two parts upon photolysis by a UV flash, which triggers the release of calcium in the sample under study. The time resolution is only limited by the intrinsic reaction rates of the cage and diffusion to the protein. The interpretation of the spectra is complicated by the fact that the spectral features of the caged compound itself change after photolysis and overlap with some protein bands of interest. Using caged Ca 2+ , ATP, or ADP, function-related events on a molecular level in the sarcoplasmic reticulum Ca 2+ -ATPase were analyzed by time-resolved Fourier transform infrared difference spectroscopy (25–27). 4. Notes 1. It should be noted that commercial suppliers of FTIR spectrometers provide instruments that plot spectra either with increasing or decreasing wavenumber. 2. The potential problems that can arise when using CaF2 cells in infrared measurements of calcium-binding proteins have been descrived in detail elsewhere (28). 3. Altering the solvent from H2O to D2O needs to be considered carefully as a potential experimental variable. Before an IR experiment is done, it is often advisable to completely exchange all protein amide groups, so that no further changes occur in the amide II band in the course of the measurement. Some internal backbone NH protons in certain proteins can exchange rather slowly and may require extended incubation in D2O at room temperature, or treatment at higher temperature, or other extreme conditions (e.g., higher pH). Nuclear magnetic resonance spectroscopy measurements indicate that at 10 C below the proteins denaturation temperature hydrogen exchange is usually very rapid, while minimizing protein denaturation (see also Subheading 3.2.1.). Note that amide hydrogen exchange is markedly dependent on pH, with low pH slowing down the reaction.
72 Fabian and Vogel 4. ATR-FTIR spectroscopy is, of course, an important technique for studying the association of membrane proteins or peptides with membranous or membrane minetic surfaces. The technique allows detection of the changes in secondary structure that accompany their binding; moreover the orientation (e.g., parallel or perpendicular to the membrane surface) can be determined. (e.g., ref. 29). 5. Protein concentrations are most-accurately determined spectroscopically using published extinction coefficients or by quantitative amino acid analysis of stock solutions. 6. The trifluoro acetate has a strong infrared absorption band at 1673 cm –1 that can overlap with the amide I band of the peptide. In some synthetic peptide preparations, it is best to remove the TFA completely. This can be accomplished by repeated lyophilization from 10 mM hydrochloric acid (30). References 1. Goormaghtigh, E., Cabiaux, V., and Ruyschaert, J.-M. (1994) Determination of soluble and membrane protein structure by Fourier transform infrared spectroscopy, in Subcellular Biochemistry, vol. 23, Physicochemical Methods in the Study of Biomembranes (Hilderson, H. J. and Ralston, B. G., eds.), Plenum, New York, pp. 329–450. 2. Fabian, H. and Mantsch, H. H. (1995) Ribonuclease A revisited: Infrared spectroscopic evidence for the lack of native-like structures in the thermally denatured state. Biochemistry 33, 10,725–10,730. 3. Alben, J. O. and Fiamingo, F. G. (1984) Fourier transform infrared spectroscopy, in Optical Techniques in Biological Research (Rousseau, D. L., ed.), Academic, New York, pp. 133–179. 4. Venyaminov, S. Y. and Prendergast, F. G. (1997) Water (H2O and D2O) molar absorptivity in the 1000–4000 cm –1 range and quantitative infrared spectroscopy of aqueous solutions. Anal. Biochem. 248, 234–245. 5. Jackson, M. and Mantsch, H. H. (1995) The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit. Rev. Biochem. Mol. Biol. 30, 95–120. 6. Nara, M., Tasumi, M., Tanokura, M., Hiraoki, T., Yazuwa, M., and Tsutsumi, A. (1994) Infrared studies of interaction between metal ions and Ca2+ -binding proteins. Marker bands for identifying the types of coordination of the side-chain COO- groups to metal ions in pike parvalbumin. FEBS Lett. 349, 84–88. 7. Nara, M., Tanokura, M., Yamamoto, T., and Tasumi, M. (1995) A comparative study of the binding effects of Mg2+ , Ca2+ , Sr2+ , and Cd2+ on calmodulin by Fouriertransform infrared spectroscopy. Biospectroscopy 1, 47–54. 8. Moffatt, D. J. and Mantsch, H. H. (1992) Fourier resolution enhancement of infrared spectral data. Methods Enzymol. 210, 192–200. 9. Surewicz, W. K., Mantsch, H. H., and Chapman, D. (1993) Determination of protein secondary structure by Fourier transform infrared spectroscopy. Biochemistry 32, 389–394.
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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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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
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Page 53 and 54: 34 Haiech and Kilhoffer Calmodulin
Page 55 and 56: 36 Haiech and Kilhoffer Fig. 3. Mec
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Page 61 and 62: 42 Haiech and Kilhoffer 62. Becking
Page 63 and 64: 44 Martin and Bayley Fig. 1. Absorp
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Page 67 and 68: 48 Martin and Bayley 4. Set the tem
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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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Page 103 and 104: 84 Table 1 Advanced Fluorescence Me
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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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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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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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