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10544221345
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m = 45° ± 1 °C) indicates that although ionic interactions do play a moderate role in stabilizing the structure of the pentameric bundle, they may be especially important in guiding the correct assembly of the helices.
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15
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10544242664
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note
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2 per residue). This results from the larger axial pore diameter in COMP, which makes residues at positions a and d more accessible to water molecules, whereas in the tetramer (1), water is excluded by the small channel radius of 1.0 to 1.3 Å. Water in the narrow pentameric channel cannot form clathrate cages around hydrophobic groups, as it does in bulk solvent (18). The resulting entropy gain may explain why a pore of the size found in the COMP pentamer does not destabilize the structure. Larger hydrophobic pores would be entropy-opposed, making hexameric or larger coiled-coil structures less probable.
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16
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10544256305
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note
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- is the most likely anion, because crystalization was performed in the presance 0.5 M NaCl.
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17
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28
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10544229067
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note
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66 (M66A) mutant to decrease the number of heavy atoms in the asymmetric unit and to Obtain an additional derfivative. Several diffraction data sets were cofected in the laboratory, as described above, and others were Collected on the BW7B beamiine at the Eurpean Molecular Biology Laboratory outstation, Hamburg, Germany (λ = 0.882 Å) and on the Swiss Norwegian beamline at the European synchroton Radiation Facility, Grenoble, France (λ = 0.875Å). Although an amino acid analysis indicated that selenomethionine was successfully incorporated in both wild-type and M66A COMP, we were not able to detect any signal from the Se atoms, using either isomorphous difference or anomalous Patterson techniques. We continued the search for heavy atom derivatives, using a traditional approach with selenomethionine M66A COMP as the native protein, because it demonstrated a higher stability of unit cell dimensions upon soaking (note the significant nonisomorphism between the native and M66A data). This search produced two useful derivatives. Alternatively, we attempted to make use of a putative axial hydrophobic cavity in the model pentameric coiled coil by preparing the xenon derivative. After subjecting native crystals to a xenon pressure between 2 and 5 bar, only weak binding was detected. However, with an improved system, we were able to apply pressure of 10 bar to the M66A mutant crystal in a standard 0.5-mm glass capillary, which resulted in a useful derivative.
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84889120137
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_, J.-Y. Zou, S. W, Cowan, M. Kjeldgaard, Acta Crystallogr. A 47, 110 (1991).
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10544240472
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note
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72.
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37
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10544254372
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note
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Supported in part by Swiss National Science Foundation grant 31-32251.91 to J.E. We thank J. N. Jansonius for support and for providing access to x-ray equipment, and also for valuable comments on the manuscript. Coordinates have been deposited in the Brookhaven Protein Bank (entry code, 1VDF).
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