Designing better diffracting crystals of biotin carboxyl carrier protein fromPyrococcus horikoshiiby a mutation based on the crystal-packing propensity of amino acids
| Autor: | Koshiro Nakai, Kentaro Tomii, Yoshinori Fukasawa, Yoshinori Matsuura, Naoki Kunishima, Kazunori D. Yamada, Hisashi Naitow |
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| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine Steric effects Protein Conformation Stereochemistry Archaeal Proteins Mutant Biotin carboxyl carrier protein Crystallography X-Ray 010403 inorganic & nuclear chemistry 01 natural sciences Protein Structure Secondary law.invention 03 medical and health sciences Pyrococcus horikoshii Structural Biology law protein crystallography Fatty Acid Synthase Type II surface engineering Amino Acids Crystallization crystal contact engineering chemistry.chemical_classification biology Chemistry Wild type biology.organism_classification Research Papers X-ray diffraction 0104 chemical sciences Amino acid Crystallography 030104 developmental biology Mutagenesis Site-Directed biology.protein Protein crystallization crystal packing Acetyl-CoA Carboxylase |
| Zdroj: | Acta Crystallographica. Section D, Structural Biology |
| ISSN: | 2059-7983 |
| Popis: | In order to improve the efficiency of protein crystallization, an alternative approach using the mutation of surface residues was devised based on the results of a statistical analysis of the crystal-packing propensity of amino acids. A systematic crystallization experiment validated the results of the statistical analysis. An alternative rational approach to improve protein crystals by using single-site mutation of surface residues is proposed based on the results of a statistical analysis using a compiled data set of 918 independent crystal structures, thereby reflecting not only the entropic effect but also other effects upon protein crystallization. This analysis reveals a clear difference in the crystal-packing propensity of amino acids depending on the secondary-structural class. To verify this result, a systematic crystallization experiment was performed with the biotin carboxyl carrier protein from Pyrococcus horikoshii OT3 (PhBCCP). Six single-site mutations were examined: Ala138 on the surface of a β-sheet was mutated to Ile, Tyr, Arg, Gln, Val and Lys. In agreement with prediction, it was observed that the two mutants (A138I and A138Y) harbouring the residues with the highest crystal-packing propensities for β-sheet at position 138 provided better crystallization scores relative to those of other constructs, including the wild type, and that the crystal-packing propensity for β-sheet provided the best correlation with the ratio of obtaining crystals. Two new crystal forms of these mutants were obtained that diffracted to high resolution, generating novel packing interfaces with the mutated residues (Ile/Tyr). The mutations introduced did not affect the overall structures, indicating that a β-sheet can accommodate a successful mutation if it is carefully selected so as to avoid intramolecular steric hindrance. A significant negative correlation between the ratio of obtaining amorphous precipitate and the crystal-packing propensity was also found. |
| Databáze: | OpenAIRE |
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