Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

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Multiple Sequence Alignment 231 19 Exploring Familial Relationships Using Multiple Sequence Alignment Aalim M. Weljie and Jaap Heringa 1. Introduction Over the course of the past 30 yr, a multitude of calcium-binding proteins has been discovered that employ several unique structural motifs for calciumion binding. The first prominent family identified bound calcium via a helixloop-helix structural motif, and was coined the EF-hand binding motif, as it occurs between the E and F helices of carp parvalbumin (1). Today, the EF-hand calcium-binding family is ubiquitous, with members implicated in varied roles such as calcium signaling cell response and calcium storage. More recently, other calcium-binding motifs such as those found in annexin repeats (2), C2 domain proteins (3), and EGF domain proteins (4) have been identified. Table 1 summarizes the characteristic amino acid sequence properties of each of these domains as provided in the PROSITE protein motif recognition database (5). Within each of these four families, there is a varying amount of primary sequence diversity that can be tolerated while maintaining calcium-binding properties. In addition, these motifs are often found within proteins as subunits, in conjunction with other functional protein domains. Such functionality is especially prevalent in eukaryotic EF-hand and C2-domain families, which often use calcium-binding in response to intracellular calcium-ion cascades as a means of cell signaling. Given the vast number of known sequences and diversity of these protein families, the obvious and popular method to make sense of the data has been to examine these proteins via sequence alignments. Much effort has been given to classification and grouping of each of these families into subfamilies and/or ancestral relationships. In particular, the EF family has been subject to exten- 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 231
232 Weljie and Heringa Table 1 Amino Acid Motifs for the Common Ca2+ -Binding Families Family Motif PROSITE Accession EF-hand D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]- {GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]- [LIVMFYW] PS00018 C2 domain [ACG]-x(2)-L-x(2,3)-d-x(1,2)-[NGSTLIF]-[GTMR]x-[STAP]-d-[PA]-[FY] PS00499 Annexin [TG]-[STV]-x(8)-[LIVMF]-x(2)-R-x(3)-[DEQNH]x(7)-[IFY]-x(7)-[LIVMF]-x(3)-[LIVMF]-x(11)- [LIVMFA]-x(2)-[LIVMF] PS00223 EGF [DEQN]-x-[DEQN](2)-C-x(3,14)-C-x(3,7)-C-x- [DN]-x(4)-[FY]-x-C PS01187 sive analysis based on primary protein sequence (6–8), nucleic acid sequence (9), and other evolutionary (10) information. Also the annexins (11,12), C2 domain families (3), and EGF family (4) have previously been analyzed. A researcher interested in a calcium-binding protein might have a number of different possible motivations to perform their own sequence alignments. For a novel protein, one may be interested to find out what sequence characteristics their protein shares with previously examined molecules and the degree of similarity to those other proteins or subfamilies. Apart from being a powerful tool to study primary sequence characteristics, sequence alignments can also be used to garner more complex information via secondary and tertiary structure predictions (13). In fact, the currently most sensitive methods in secondary structure prediction all rely on a multiple alignment as input. Also, evolutionary relationships and phylogenetic trees are normally established from reliable sequence alignments. Such avenues of analysis are particularly important, given the rate at which sequence information is becoming available, resulting in an ever-increasing gap between sequence and structure information. Whereas much research has been concentrated on the analysis of sequence alignments, the most critical aspect of any such examination remains the quality of the alignment itself. An erroneous alignment will produce erroneous results and will also confuse the aforementioned reliant techniques. Manual alignments utilizing expert knowledge are the ideal way to go about sequence comparisons, and for small amounts of data this is certainly the method of choice. However, the obvious challenges of dealing with large amounts of sequence data have led to the development of many algorithms that use silicon, rather than human, computation power. Most multiple alignment methods
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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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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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Page 225 and 226: 206 Clarke and Vogel cal calcium-bi
Page 227 and 228: 208 Clarke and Vogel Fig. 1. 113 Cd
Page 233 and 234: 214 Clarke and Vogel The linewidth
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Page 271 and 272: 252 Weljie and Heringa 44. Thompson
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Page 275 and 276: 256 Li et al. In order for these ex
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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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METHODS IN MOLECULAR BIOLOGY TM •
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