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Abstract Background The generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution. Existing methods, such as combinatorial active site testing and residual coupling analysis, depend primarily on the evolutionary conserved information to find the hotspot residues. Hardly any attention has been paid to another important functional and structural determinants, the functionally correlated variation information--coevolution. Results In this paper, we suggest a new method, named combinatorial coevolving-site saturation mutagenesis (CCSM), in which the functionally correlated variation sites of proteins are chosen as the hotspot sites to construct focused mutant libraries. The CCSM approach was used to improve the thermal stability of α-amylase from Bacillus subtilis CN7 (Amy7C). The results indicate that the CCSM can identify novel beneficial mutation sites, and enhance the thermal stability of wild-type Amy7C by 8°C (T5030), which could not be achieved with the ordinarily rational introduction of single or a double point mutation. Conclusions Our method is able to produce more thermostable mutant α-amylases with novel beneficial mutations at new sites. It is also verified that the coevolving sites can be used as the hotspots to construct focused mutant libraries in protein engineering. This study throws new light on the active researches of the molecular coevolution.

Storage of hydrogen gas (H2) in clathrate hydrate at ambient conditions has many potential applications, such as clean energy, environment and ecology protection, submarine, and space rocket. In this research article, the hydrogen gas hydrate is studied by using higher level quantum chemical methods such as MP2, CCSD, and CCSD(T). The interaction energy terms in the hydrate cage and the guest molecular cluster are calculated and analyzed. Some useful conclusion points are summarized as follows: (1) The capacity and stability of the hydrogen molecular clusters in hydrate cages are determined by three energy terms: the hydrogen bond energy (ΔEH-b) of water molecules, the interaction energy (ΔEcage-clst) between the hydrate cage and the guest molecular cluster, and the interaction energy (ΔEclst) of the guest molecular cluster in the cage. (2) The energy term ΔEclst of the hydrogen molecular cluster in the hydrate cage is the only repulsive energy contribution (positive values) to the stability of the gas-hydrate cages, which increases with the number of hydrogen molecules and decreases with the size of the cage volume. (3) The strong hydrogen bond energy ΔEH-b remains constant in a broad range of 2.74 Å to 2.86 Å, in which the change in ΔEH-b is less than ±5 kJmol−1. (4) Capacity of hydrogen molecules in the hydrate cage depends on the volume of the hydrate cages. When the side length RO-O is 2.82 Å, at most 4 hydrogen molecules can be stored in the 512 cage. In large hydrate cages (51262 and 51264), more hydrogen molecules can be stored. [ABSTRACT FROM AUTHOR]

New developments of the 3d-RISM-HNC integral equation for describing the solvation properties of complex molecules in water are discussed. A well-calibrated 3d-RISM-HNC integral equation has the potential to act as a valuable intermediate approach.

According to the "distorted key" theory as elaborated in a review article years ago (Chou, K.C.: Analytical Biochemistry, 1996, 233, 1-14), the knowledge of the cleavable peptides by SARS-CoV M(pro) (severe acute respiratory syndrome coronavirus main proteinase) can provide very useful insights on developing drugs against SARS. In view of this, the softwares, ZCURVE_CoV 1.0 and ZCURVE_CoV 2.0 (http://tubic.tju.edu.cn/sars/), developed recently for SARS-Coronavirus are used to analyze the 36 complete SARS-Coronavirus RNA sequences in the gene bank NCBI (http://www.ncbi.nlm.nih.gov/) from different sources for protein coding genes, and to search for the cleavage sites of SARS-CoV M(pro) in polyproteins pp1a and pp1ab. A total of 396 cleavage points are found in the 36 SARS-Coronavirus and 11 cleavable octapeptides abstracted from the 396 cleavage sites. The statistical distributions of amino acids for the cleavable octapeptides at the subsites R4, R3, R2, R1, R1', R2', R3' and R4' are calculated. The cleavage-specific positions are on R2, R1 and R1', and the positions R3 and R4 are featured by some certain specificity for SARS-CoV M(pro). The structural characters of amino acid residues around the cleavage-specific positions are discussed. Two most promising octapeptides, i.e., NH(2)-ATLQ downward arrowAIAS-COOH and NH(2)-ATLQ downward arrowAENV-COOH, are selected to be the candidates for chemical modification, converting into the inhibitors of SARS-CoV M(pro). A possible strategy to convert a cleavable octapeptide by SARS enzyme into a drug candidate against SARS is elucidated.