Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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MALLS and Sedimentation Equilibrium 127 11 Multiangle Laser Light Scattering and Sedimentation Equilibrium Leslie D. Hicks, Jean-René Alattia, Mitsuhiko Ikura, and Cyril M. Kay 1. Introduction Multiangle laser light scattering (MALLS) and sedimentation equilibrium are two powerful techniques used to characterize the association properties of proteins and their interactions in solution under physiological conditions. Both techniques have undergone a resurgence as a result of the advent of recombinant technologies which has enabled the generation of reasonable quantities of biologically significant proteins that exist in vivo in small amounts so that they can now be characterized physicochemically. As well, new technical developments with both techniques have made them much more sensitive and user friendly. In the case of static light scattering, this includes the use of lasers and modern detectors on-line with size exclusion chromatography so that one can establish absolute molecular weights of individual protein fractions eluting from the column. With sedimentation equilibrium, the Optima XL-I centrifuge (developed by Beckman, Palo Alto, CA) is equipped with both a new photoelectric scanning absorption optical system enabling exact measurement of concentration profiles at wavelengths of 190–800 nm and an interference optical system allowing the measurement of much higher concentration gradients. In both cases, powerful computer programs have been developed for data evaluation. Examples of the use of both techniques to study the association properties of cadherin in the presence and absence of calcium are described later. 2. MALLS 2.1. Instrument Set-Up and Preparation 1. The instrumental setup consists sequentially of a solvent reservoir; Shimadzu model DGU-4A vacuum solvent degasser; Waters model 510 HPLC pump From: Methods in Molecular Biology, vol. 173: Calcium-Binding Protein Protocols, Vol. 2: Methods and Techniques Edited by: H. J. Vogel © Humana Press Inc., Totowa, NJ 127
128 Hicks et al. equipped with a pulse dampener, in-line 0.2-µm and 0.1-µm membrane filters, and a Rheodyne Model 7125 sample injector complete with a 1.0-mL sample loop; Pharmacia Superose 12 FPLC Gel Filtration column; Wyatt Technology Corporation Dawn F Multi-Angle Laser Light Scattering Photometer equipped with a 10-mW 488-nm argon-ion laser and temperature-controlled flow cell and read head; Wyatt Technology Corporation Optilab Model 903 Differential Refractometer equipped with temperature-controlled 1-mm path length flow cell; and finally, a Lauda model K-2/R circulating water bath with insulated hoses connected to both the Dawn F and Optilab 903. 2. Periodically, the 90° detector in the Dawn F is calibrated with HPLC grade Toluene, which has a known scattering intensity, or Rayleigh Ratio, R Θ, following the procedures outlined in the Dawn F instrument manual (1). The Optilab 903 is calibrated with a solution prepared from ultrapure, anhydrous, NaCl following the procedures outlined in the Optilab 903 instrument manual (2). 3. The desired run temperature (usually 20°C.) is set for both the Dawn F and Optilab 903 using the temperature control setting on the Dawn F and on the Lauda water bath at least 24 h prior to calibrating or running samples to allow the Optilab signal (DRI) to stabilize, because it is very sensitive to temperature changes. As well, the Optilab lamp is powered up for a minimum of 24 h before data collection to reduce signal drift over time. 4. Prior to pumping through the instrument setup, the buffer solution is filtered through 0.1-µm membrane filters. A minimum volume of 100–200 mL is then pumped through the set-up at a maximum of 1.0 mL/min to allow the column to equilibrate and the Optilab signal to stabilize. 2.2. Sample Preparation 1. The protein solution (typically 400–500 µL of 1–5 mg/mL solution) is dialyzed against the buffer solution for 24 h and then filtered through a 0.22-µm syringe filter prior to injecting into the instrument set-up. After filtering, the sample solution is spun for 3–5 min in a microfuge to remove minute air bubbles introduced in the filtering process. 2.3. Data Collection 1. Prior to running a sample, the intensity of the scattering signal at different angles is normalized to the intensity of the signal at 90° in the Dawn F using a solution of BSA (Sigma 98% monomer), prepared in the same manner as the sample solution. With the HPLC pump operating at a flow rate of 0.8 mL/min, which provides reasonable separation of peaks and relatively quiet MALLS signals in reasonably short run times, a 40-µL aliquot of BSA solution (approx 5 mg/mL) is injected into the system using the Rheodyene injector. Data collection is performed using Wyatt Technology’s Astra software. 2. Once the MALLS and DRI signals have stabilized after elution of the BSA peaks, an aliquot of the sample solution is injected (with the Superose 12 column and a sensitivity setting of 100, an injected mass of approx 0.25 mg will produce a full-
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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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Page 103 and 104: 84 Table 1 Advanced Fluorescence Me
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Page 109 and 110: 90 Johnson and Tikunova is removed
Page 111 and 112: 92 Johnson and Tikunova function of
Page 113 and 114: 94 Johnson and Tikunova Fig. 2. 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
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Page 143 and 144: 124 Lopez and Makhatadze 2.5 mL are
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Page 157 and 158: 138 Trewhella and Krueger This chap
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Page 161 and 162: 142 Trewhella and Krueger Table 1 C
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Page 165 and 166: 146 Trewhella and Krueger At very l
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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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