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Elongation factor G (EF-G) is a G protein factor that catalyzes the translocation step in protein synthesis on the ribosome. Its GTP conformation in the absence of the ribosome is currently unknown. We present the structure of a mutant EF-G (T84A) in complex with the non-hydrolysable GTP analogue GDPNP. The crystal structure provides a first insight into conformational changes induced in EF-G by GTP. Comparison of this structure with that of EF-G in complex with GDP suggests that the GTP and GDP conformations in solution are very similar and that the major contribution to the active GTPase conformation, which is quite different, therefore comes from its interaction with the ribosome.

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A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T(1/2) of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated higher thermostability than its wild type predecessor. To explore the structure-function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme by 2-folds when compared with WT. The mutant enzyme showed enhanced T(1/2) of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher affinity for substrate (decreased K ( m )) in comparison to the wild type. The k (cat) and catalytic efficiency (k (cat)/K ( m )) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a solvent exposed hydrophobic residue (Ile) in WT with a hydrophilic residue (Thr) in mutant might impart thermostability to the protein structure.