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Journal Articles

"Crystal lattice engineering", an approach to engineer protein crystal contacts by creating intermolecular symmetry; Crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

Yamada, Hidenori*; Tamada, Taro; Kosaka, Megumi*; Miyata, Kohei*; Fujiki, Shinya*; Tano, Masaru*; Moriya, Masayuki*; Yamanishi, Mamoru*; Honjo, Eijiro; Tada, Horiko*; et al.

Protein Science, 16(7), p.1389 - 1397, 2007/07

 Times Cited Count:39 Percentile:60.19(Biochemistry & Molecular Biology)

In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To further characterize the role of the introduced leucine residues in crystallization of RNase 1, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized, and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intra molecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Oral presentation

The Contribution of the Japanese team; Neutron diffraction experiment of fully deuterated proteins, such as RNase A, insulin, myoglobin and so on

Niimura, Nobuo*; Tanaka, Ichiro*; Onishi, Yuki*; Ostermann, A.*; Kurihara, Kazuo; Honjo, Eijiro; Kuroki, Ryota; Futami, Junichiro*; Yamada, Hidenori*

no journal, , 

Our goal is to develop new methods for determining macromolecular crystal structures ${it ab initio}$ from neutron diffraction data and to validate these methods by making applications to several proteins. The proposed methods, which have the potential to be more powerful than X-ray ones, exploit perfectly isomorphous pairs of crystals that differ by replacement of D atoms with H atoms in selected amino-acid residues. This research involves collaboration among teams on three continents that have expertise in different aspects of neutron crystallography. The Japanese team measures high-resolution neutron diffraction data from protein crystals to answer questions about H bonding, protonation, hydration, and enzyme mechanisms. The team will focus on fundamental and simple proteins such as myoglobin, insulin, and RNase A at the first stage and then on rubredoxin, aldose reductase and other samples from the French team. In the meeting the research plan of the Japanese team will be reported.

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