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The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes how the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia take place, and how these interactions are inhibited by LMWH and DP4. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

ST8Sia II (STX) is a highly homologous mammalian polysialyltransferase (polyST), which is a validated tumor-target in the treatment of cancer metastasis reliant on tumor cell polysialylation. PolyST catalyzes the synthesis of α2,8-polysialic acid (polySia) glycans by carrying out the activated CMP-Neu5Ac (Sia) to N- and O-linked oligosaccharide chains on acceptor glycoproteins. In this review article, we summarized the recent studies about intrinsic correlation of two polybasic domains, Polysialyltransferase domain (PSTD) and Polybasic region (PBR) within ST8Sia II molecule, and suggested that the critical amino acid residues within the PSTD and PBR motifs of ST8Sia II for polysialylation of Neural cell adhesion molecules (NCAM) are related to ST8Sia II activity. In addition, the conformational changes of the PSTD domain due to point mutations in the PBR or PSTD domain verified an intramolecular interaction between the PBR and the PSTD. These findings have been incorporated into Zhou’s NCAM polysialylation/cell migration model, which will provide new perspectives on drug research and development related to the tumor-target ST8Sia II.

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

Acetoin is an important four-carbon compound that has many applications in foods, chemical synthesis, cosmetics, cigarettes, soaps, and detergents. Its stereoisomer (S)-acetoin, a high-value chiral compound, can also be used to synthesize optically active drugs, which could enhance targeting properties and reduce side effects. Recently, considerable progress has been made in the development of biotechnological routes for (S)-acetoin production. In this review, various strategies for biological (S)- acetoin production are summarized, and their constraints and possible solutions are described. Furthermore, future prospects of biological production of (S)-acetoin are discussed.

Background: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. Objective: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. Methods: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. Results: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to β-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. Conclusion: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

Background: The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies. Objective: To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes? Methods: Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4. Results: The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4. Conclusion: The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Background: Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. Objective: In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). Methods: Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. Results: According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. Conclusion: This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

Background: The relationship between protein structure and its bioactivity is one of the fundamental problems for protein engineering and pharmaceutical design. Method: A new method, called SPTD (Simulated Protein Thermal Detection), was proposed for studying and improving the thermal stability of enzymes. The method was based on the evidence observed by conducting the MD (Molecular Dynamics) simulation for all the atoms of an enzyme vibrating from the velocity at a room temperature (e.g., 25°C) to the desired working temperature (e.g., 65°C). According to the recorded MD trajectories and the coordinate deviations of the constituent residues under the two different temperatures, some new strategies have been found that are useful for both drug delivery and starch industry. Conclusion: The SPTD technique presented in this paper may become a very useful tool for pharmaceutical design and protein engineering.

As a subset of glycosyltransferases, the family of sialyltransferases catalyze transfer of sialic acid (Sia) residues to terminal non-reducing positions on oligosaccharide chains of glycoproteins and glycolipids, utilizing CMP-Neu5Ac as the activated sugar nucleotide donor. In the four known sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), the ST8Sia family catalyzes synthesis of α2, 8-linked sialic/polysialic acid (polySia) chains according to their acceptor specificity. We have determined the 3D structural models of the ST8Sia family members, designated ST8Sia I (1), II(2), IV(4), V(5), and VI(6) using the Phyre2 server. Accuracy of these predicted models are based on the ST8Sia III crystal structure as the calculated template. The common structural features of these models are: (1) Their parallel templates and disulfide bonds are buried within the enzymes and are predominately surrounded by helices; (2) The anti-parallel β-sheets are located at the N-terminal region of the enzymes; (3) The mono-sialytransferases (mono-STs), ST8Sia I and ST8Sia VI, contain only a single pair of disulfide bonds, and there are no anti-parallel β-sheets in ST8Sia VI; (4) The Nterminal region of all of the mono-STs are located some distant away from their core structure; (5) These conformational features show that the 3D structures of the mono-STs are less compact than the two polySTs, ST8Sia II and ST8Sia IV, and the oligo-ST, ST8Sia III. These structural features relate to the catalytic specificity of the monoSTs; (6) In contrast, the more compact structural features of ST8Sia II, ST8Sia IV and ST8Sia III relate to their ability to catalyze the processive synthesis of oligo- (ST8Sia III) and polySia chains (ST8Sia II & ST8Sia IV); (7) Although ST8Sia II, III and IV have similar conformations in their corresponding polysialyltransferase domain (PSTD) and polybasic region (PBR) motifs, the structure of ST8Sia III is less compact than ST8Sia II and ST8Sia IV, and the amino acid components of the several three-residue-loops in the two motifs of ST8Sia III are different from that in ST8Sia II and ST8Sia IV. This is likely the structural basis for why ST8Sia III is an oligoST and not able to polysialylate and; (8) In contrast, essentially all amino acids within the threeresidue- loops in the PSTD of ST8Sia II and ST8Sia IV are highly conserved, and many amino acids in the loops and the helices of these two motifs are critical for NCAM polysialylation, as determined by mutational analysis and confirmed by our recent NMR results. In summary, these new findings provide further insights into the molecular mechanisms underlying polyST-NCAM recognition, polySTpolySia/ oligoSia interactions, and polysialylation of NCAM.

(2R,3R)-2,3-Butanediol has many industrial applications, such as it is used as an antifreeze agent and low freezing point fuel. In addition, it is particularly important to provide chiral groups in drugs. In recent years, this valuable bio-based chemical has attracted increasing attention, and significant progress has been made in the development of microbial cell factories for (2R,3R)-2,3-butanediol production. This article reviews recent advances and challenges in microbial routes to (2R,3R)-2,3- butanediol production, and highlights the metabolic engineering and synthetic biological approaches used to improve titers, yields, productivities, and optical purities. Finally, a systematic and integrative strategy for developing high-performance microbial cell factories is proposed.

All residues in an alpha helix can be characterized and dispositioned on a 2D the wenxiang diagram, which possesses the following features: (1) the relative locations of the amino acids in the -helix can be clearly displayed regardless how long it is; (2) direction of an alphahelix can be indicated; and (3) more information regarding each of the constituent amino acid residues in an alpha helix. Owing to its intuitionism and easy visibility, wenxiang diagrams have had an immense influence on our understanding of protein structure, protein-protein interactions, and the effect of helical structural stability on protein conformational transitions. In this review, we summarize two recent applications of wenxiang diagrams incorporating NMR spectroscopy in the researches of the coiled-coil protein interactions related to the regulation of contraction or relaxation states of vascular smooth muscle cells, and the effects of α-helical stability on the protein misfolding in prion disease, in hopes that the gained valuable information through these studies can stimulate more and more widely applications of wenxiang diagrams in structural biology.

Although not being classified as the most fundamental protein structural elements like α-helices and β-strands, the loop segment may play considerable roles for protein stability, flexibility, and dynamic activity. Meanwhile, the protein loop is also quite elusive; i.e. its interactions with the other parts of protein as well as its own shape-maintaining forces have still remained as a puzzle or at least not quite clear yet. Here, we report a molecular force, the so-called polar hydrogen–πinteraction (Hp–π), which may play an important role in supporting the backbones of protein loops. By conducting the potential energy surface scanning calculations on the quasi π-plane of peptide bond unit, we have observed the following intriguing phenomena: (1) when the polar hydrogen atom of a peptide unit is perpendicularly pointing to the π-plane of other peptide bond units, a remarkable Hp–πinteraction occurs; (2) the interaction is distance and orientation dependent, acting in a broad space, and belonging to the ‘point-to-plane’ one. The molecular force reported here may provide useful interaction concepts and insights into better understanding the loop’s unique stability and flexibility feature, as well as the driving force of the protein global folding.

Due to the low toxicity, easy synthesis, rapid elimination, and less side effect, more and more peptide inhibitors are emerging as the effective drugs that are clinically used in therapies of a number of diseases. At the same time the computer-aided drug design (CADD) methods have remarkably developed. In this mini review the newly developed peptide inhibitors and drugs are introduced, including peptide vaccines for cancers, peptide inhibitors for HIV, Alzheimer's disease and related diseases, and the peptides as the leading compounds of drugs. The recent progress in the theory and methodology of peptide inhibitor design is reviewed. (1) The flexible protein-peptide docking model is introduced, in which the peptide structures are treated as segment-flexible chains using genetic algorithm and special force field parameters. (2) The 'Wenxiang diagram' is illustrated for protein-peptide interaction analysis that has been successfully used in the coiled-coil interaction analysis. (3) The 'Distorted key' theory is reviewed, which is an effective method to convert the peptide inhibitors to the small chemical drugs. (4) The amino acid property-based peptide prediction method (AABPP) is described that is a twolevel QSAR prediction network for the bioactivity prediction of peptide inhibitors. (5) Finally, several types of molecular interactions between protein and peptide ligands are summarized, including cation-π interactions; polar hydrogen-π interactions; and π-π stocking interactions.

Synthesis of 2,8-polysialic acid (polySia) glycans are catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), which are two members of the ST8Sia gene family of sialytransferases. During polysialylation, both STX and PST catalyze the transfer of multiple Sia residues from the activated sugar nucleotide precursor, CMP-Neu5Ac (Sia), to terminal Sia residues on N- and Olinked oligosaccharide chains on acceptor glycoproteins, including the neural cell adhesion molecule (NCAM), which is the major carrier protein of polySia. Based on our new findings and previously published studies, this review summarizes the present concepts regarding the molecular mechanism underlying regulation of protein-specific polysialylation of NCAM that includes the following: (1) Determination of the catalytic domains and specific regions within ST8Sia IV for recognizing and catalyzing the efficient polysialylation of NCAM; (2) Identification of key amino acid residues within the PSTD motif of ST8Sia IV that are essential for polysialylation; (3) Verification of key amino acids in the PBR domain of ST8Sia IV required for NCAM-specific polysialylation; and (4) a 3D conformational study of ST8Sia IV based on the Phyre2 server to discover the relationship between the structure and its functional domains of the polyST. Based on these results, our 3D model of ST8Sia IV was used to identify and characterize the catalytic domains and amino acid residues critical for catalyzing polysialylation, and have provided new structural information for supporting a detailed mechanism of polyST-NCAM interaction required for polysialylation of NCAM, findings that have not been previously reported.

This paper presents a new approach for obstacle avoidance of small mobile robots, which combine the position sensitive detector (PSD) with digital compass. It is important for an autonomous robot to explore its surroundings in performing the task of localization and navigation for searching. Because of the complexity of the environment, one simple kind of sensors is not sufficient for robot to accomplish these tasks. In this paper, the small mobile robots are enabled to identify barriers and distinguish surroundings by using the angle signal from the digital compass which is generally mounted on the robot. Experimental results indicate that this approach based on digital compass shows great potential in autonomous robot obstacle avoidance.

In drug design and enzyme engineering, the information of interactions between receptors and ligands is crucially important. In many cases, the protein structures and drug-target complex structures are determined by a delicate balance of several weak molecular interaction types. Among these interaction forces several unconventional interactions play important roles, however, less familiar for researchers. The cation-π interaction is a unique noncovalent interaction only acting between aromatic amino acids and organic cations (protonated amino acids) and inorganic cations (proton and metallic). This article reports new study results in the interaction strength, the behaviors and the structural characters of cation-π interactions between aromatic amino acids (Phe, Tyr, and Trp) and organic and inorganic cations (Lys, Arg, H, H3O, Li, Na, K, Ca2, and Zn2) in gas phase and in solutions (water, acetonitrile, and cyclohexane). Systematical research revealed that the cation-π interactions are point-to-plane (aromatic group) interactions, distance and orientationdependent, and the interaction energies change in a broad range. In gas phase the cation-π interaction energies between aromatic amino acids (Phe, Tyr, and Trp) and metallic cations (Li, Na, K, Ca2, and Zn2) are in the range -12 to -160 kcal/mol, and the interaction energies of protonated amino acids (Arg and Lys) are in the range from -9 to -18 kcal/mol. In solutions the cation-π energies decrease with the dielectric constant of solvents. However, in aqueous solution the cation-π energies of H3O and protonated amino acids are less affected by solvation effects. The applications of unconventional interaction forces in drug design and in protein engineering are introduced.

Transmissible spongiform encephalopathies (TSEs) are prion protein misfolding diseases that involve the accumulation of an abnormal -sheet-rich prion protein aggregated form (PrPsc) of the normal - helix-rich prion protein (PrPc) within the central nervous system (CNS) and other organs. On account of its large size and insolubility properties, characterization of PrPsc is quite difficult. A soluble intermediate, called PrP or o, exhibiting many of the same features as PrPsc, can be generated using a combination of low pH and/or mild denaturing conditions. Here, we review the current knowledge on the following five issues relevant to the conversion mechanisms of PrPc to PrPsc : (1) How is the Stability of the Helical Structures in the Native PrPc Related to the Primary Structure of the PrPc (2) Why the Low pH Solution System is a Ideal Trigger of PrPc to PrPsc Conversion (3) How are the Structural and Dynamical Characteristics of the -helixrich Intermediates Determined using NMR Data (4) How are the Premolten (PrP4 and PrP) and -Oligomer (PrP) Intermediates Detected and Assayed, and (5) Can the Disordered N-terminal Domain be folded into the Structural Segment? Particularly, Chous wenxiang diagram (http://en.wikipedia.org/wiki/Wenxiang_diagram) was introduced for providing an intuitive picture. This review may help to further understand the prion protein misfolding mechanism.

For quite a long period of time in history, many intense efforts have been made to determine the 3D (threedimensional) structure of the M2 proton channel. The reason why the M2 proton channel has attracted so many attentions is because (1) it is the key for really understanding the life cycle of influenza viruses, and (2) it is indispensable for conducting rational drug design against the flu viruses. Recently, the long-sough 3D structures of the M2 proton channels for both influenza A and B viruses were consecutively successfully determined by the high-resolution NMR spectroscopy (Schnell J.R. and Chou, J.J., Nature, 2008, 451: 591-595; Wang, J., Pielak, R.M., McClintock, M.A., and Chou, J.J., Nature Structural & Molecular Biology, 2009,16: 1267-1271). Such a milestone work has provided a solid structural basis for in-depth understanding the action mechanism of the M2 channel and rationally designing effective drugs against influenza viruses. This review is devoted to, with the focus on the M2 proton channel of influenza A, addressing a series of relevant problems, such as how to correctly understand the novel allosteric inhibition mechanism inferred from the NMR structure that is completely different from the traditional view, what the possible impacts are to the previous functional studies in this area, and what kind of new strategy can be stimulated for drug development against influenza.

1, 3-propanediol (1, 3-PD) is biologically synthesized by glycerol dehydratase (GDHt) and 1, 3-propanediol dehydrogenase (PDOR). In present study, the gldABC gene, encoding GDHt from Klebsiella pneumoniae and the yqhD gene, encoding PDOR isoenzyme from E.coli BL21 were cloned and co-expressed in E.coli JM109 using plasmid pSE380. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration to study the properties. Optimal temperature and pH of recombinant GDHt with specific activity of 85.8 U/mg were 45 °C and 9.0; and optimal temperature and pH of recombinant YqhD with specific activity of 80.0 U/mg were 37 °C, 7.0. The microbial conversion of 1,3-PD from glycerol by this recombinant E. coli strain was studied and the production of 1,3-PD was about 28.0 g/l.

Introduction:The 2009-H1N1 influenza pandemic has prompted new global efforts to develop new drugs and drug design techniques to combat influenza viruses. While there have been a number of attempts to provide drugs to treat influenza, drug resistance has been a major problem with only four drugs currently approved by the FDA for its treatment.Areas covered:In this review, the drug-resistant problem of influenza A viruses is discussed and summarized. The article also introduces the experimental and computational structures of drug targeting proteins, neuraminidases, and of the M2 proton channel. Furthermore, the article illustrates the latest drug candidates and techniques of computer-aided drug design with examples of their application, including virtual in silicoscreening and scoring, AutoDock and evolutionary technique AutoGrow.Expert opinion:Structure-based drug design is the inventive process for finding new drugs based on the structural knowledge of the biological target. Computer-aided drug design strategies and techniques will make drug discovery more effective and economical. It is anticipated that the recent advances in structure-based drug design techniques will greatly help scientists to develop more powerful and specific drugs to fight the next generation of influenza viruses.

Importance of the field:The recent advances in visual representations of molecular properties in 3D space are summarized, and their applications in molecular modeling study and rational drug design are introduced. The visual representation methods provide us with detailed insights into protein–ligand interactions, and hence can play a major role in elucidating the structure or reactivity of a biomolecular system.Areas covered in this review:Three newly developed computation and visualization methods for studying the physical and chemical properties of molecules are introduced, including their electrostatic potential, lipophilicity potential and excess chemical potential. The newest application examples of visual representations in structure-based rational drug are presented.What the reader will gain:The 3D electrostatic potentials, calculated using the empirical method (EM-ESP), in which the classical Coulomb equation and traditional atomic partial changes are discarded, are highly consistent with the results by the higher level quantum chemical method. The 3D lipophilicity potentials, computed by the heuristic molecular lipophilicity potential method based on the principles of quantum mechanics and statistical mechanics, are more accurate and reliable than those by using the traditional empirical methods. The 3D excess chemical potentials, derived by the reference interaction site model-hypernetted chain theory, provide a new tool for computational chemistry and molecular modeling.Take home message:For structure-based drug design, the visual representations of molecular properties will play a significant role in practical applications. It is anticipated that the new advances in computational chemistry will stimulate the development of molecular modeling methods, further enriching the visual representation techniques for rational drug design, as well as other relevant fields in life science.

This review is to summarize three new QSAR (quantitative structure-activity relationship) methods recently developed in our group and their applications for drug design. Based on more solid theoretical models and advanced mathematical techniques, the conventional QSAR technique has been recast in the following three aspects. (1) In the fragment-based two dimensional QSAR, or abbreviated as FB-QSAR, the molecular structures in a family of drug candidates are divided into several fragments according to the substitutes being investigated. The bioactivities of drug candidates are correlated with physicochemical properties of the molecular fragments through two sets of coefficients: one is for the physicochemical properties and the other for the molecular fragments. (2) In the multiple field three dimensional QSAR, or MF-3D-QSAR, more molecular potential fields are integrated into the comparative molecular field analysis (CoMFA) through two sets of coefficients: one is for the potential fields and the other for the Cartesian three dimensional grid points. (3) In the AABPP (amino acid-based peptide prediction), the bioactivities of peptides or proteins are correlated with the physicochemical properties of all or partial residues of the sequence through two sets of coefficients: one is for the physicochemical properties of amino acids and the other for the weight factors of the residues. Meanwhile, an iterative double least square (IDLS) technique is developed for solving the two sets of coefficients in a training dataset alternately and iteratively. Using the two sets of coefficients, one can predict the bioactivity of a query peptide, protein, or drug candidate. Compared with the old methods, the new QSAR approaches as summarized in this review possess machine learning ability, can remarkably enhance the prediction power, and provide more structural information. Meanwhile, the future challenge and possible development in this area have been briefly addressed as well.

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (Tm, the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.

A new open reading frame in Thermobifida fusca sequenced genome was identified to encode a new trehalose synthase, annotated as "glycosidase" in the GenBank database, by bioinformatics searching and experimental validation. The gene had a length of 1830 bp with about 65% GC content and encoded for a new trehalose synthase with 610 amino acids and deduced molecular weight of 66 kD. The high GC content seemed not to affect its good expression in E. coli BL21 in which the target protein could account for as high as 15% of the total cell proteins. The recombinant enzyme showed its optimal activities at 25 degrees and pH 6.5 when it converted substrate maltose into trehalose. However it would divert a high proportion of its substrate into glucose when the temperature was increased to 37 degrees, or when the enzyme concentration was high Its activity was not inhibited by 5 mM heavy metals such as Cu2+, Mn2+, and Zn2+ but affected by high concentration of glucose. Blasting against the database indicated that amino acid sequence of this protein had maximal 69% homology with the known trehalose synthases, and two highly conserved segments of the protein sequence were identified and their possible linkage with functions was discussed.