It is well known that protein crystallizability can be influenced by site-directed mutagenesis of residues on the molecular surface of proteins, indicating that the intermolecular interactions in crystal-packing regions may play a crucial role in the structural regularity at atomic resolution of protein crystals. Here, a systematic examination was made of the improvement in the diffraction resolution of protein crystals on introducing a single mutation of a crystal-packing residue in order to provide more favourable packing interactions, using diphthine synthase from Pyrococcus horikoshii OT3 as a model system. All of a total of 21 designed mutants at 13 different crystal-packing residues yielded almost isomorphous crystals from the same crystallization conditions as those used for the wild-type crystals, which diffracted X-rays to 2.1 A resolution. Of the 21 mutants, eight provided crystals with an improved resolution of 1.8 A or better. Thus, it has been clarified that crystal quality can be improved by introducing a suitable single mutation of a crystal-packing residue. In the improved crystals, more intimate crystal-packing interactions than those in the wild-type crystal are observed. Notably, the mutants K49R and T146R yielded crystals with outstandingly improved resolutions of 1.5 and 1.6 A, respectively, in which a large-scale rearrangement of packing interactions was unexpectedly observed despite the retention of the same isomorphous crystal form. In contrast, the mutants that provided results that were in good agreement with the designed putative structures tended to achieve only moderate improvements in resolution of up to 1.75 A. These results suggest a difficulty in the rational prediction of highly effective mutations in crystal engineering.

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and it will be helpful for designing stable mutants. In this work, we have analyzed the stability of protein mutants using three different data sets of 1791, 1396, and 2204 mutants, respectively, for thermal stability (DeltaTm), free energy change due to thermal (DeltaDeltaG), and denaturant denaturations (DeltaDeltaGH2O), obtained from the ProTherm database. We have classified the mutants into 380 possible substitutions and assigned the stability of each mutant using the information obtained with similar type of mutations. We observed that this assignment could distinguish the stabilizing and destabilizing mutants to an accuracy of 70-80% at different measures of stability. Further, we have classified the mutants based on secondary structure and solvent accessibility (ASA) and observed that the classification significantly improved the accuracy of prediction. The classification of mutants based on helix, strand, and coil distinguished the stabilizing/destabilizing mutants at an average accuracy of 82% and the correlation is 0.56; information about the location of residues at the interior, partially buried, and surface regions of a protein correctly identified the stabilizing/destabilizing residues at an average accuracy of 81% and the correlation is 0.59. The nine subclassifications based on three secondary structures and solvent accessibilities improved the accuracy of assigning stabilizing/destabilizing mutants to an accuracy of 84-89% for the three data sets. Further, the present method is able to predict the free energy change (DeltaDeltaG) upon mutations within a deviation of 0.64 kcal/mol. We suggest that this method could be used for predicting the stability of protein mutants.

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We report the unprecedented formation of ruthenium(ii) complexes containing dithiocarbazate ligand, together with their DFT and biological studies.

Noncovalent interactions involving heteroaromatic ring systems play a major role in determining the function of many chemical as well as biological molecules. Therefore, detailed quantitative analyses of the π-interactions (X-H‚‚‚π and π‚‚‚π) were carried out for nitrogen-containing π-systems (isoxazole, imidazole, and indole moieties). Statistical analysis of the geometrical properties for the oxygen, nitrogen, and carbon atom donors with the heterocyclic π-system acceptors showed that carbon donors participate in relatively large numbers for X-H‚‚‚π interactions, and they adopt T-shaped geometry. The π‚‚‚π interaction analysis was categorized into three types based on the involvement of the heterocyclic π-systems with themselves, with any other benzene ring present in that particular structure, and their influence on the formation of π-interactions between benzene-benzene rings found in that particular compound. The π-systems in all the three categories prefer to form offset stacking π‚‚‚π interaction geometry. The benzene rings which normally favor the formation of T-shaped geometry are found to prefer offset stacking geometry, which may be due to the influence of the heteroatom. The π-systems in these heterocyclic structures behave similar to the phenylalaninephenylalanine interactions in proteins, and therefore this quantitative analysis can serve as a guide for structural and biological studies.

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KR thanks Dr Babu Varghese, SAIF, IIT-Madras, India, for his help with the data collection and the management of Kandaswami Kandar's College, Velur, Namakkal, India, for their encouragement to pursue the programme. Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: BT5046). metal-organic compounds m1174 Ravichandran et al.

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