Prediction of membrane proteins in Mycobacterium tuberculosis using a support vector machine algorithm.

We report our finding of linear clustering of signal sequences at the N-terminus of M.tb membrane proteins, directing membrane localization. Although it is widely accepted that membrane proteins have signal peptides at the N-terminus, statistical ensemble analysis of Support Vector Machine prediction results indicate that M.tb membrane proteins have embedded N-terminal sequence patterns beyond the signal peptides previously identified in E. coli. The additional patterns at the N-terminus of M.tb membrane proteins may have correlations to their unique enzymatic functions and unusual characteristics such as membrane interaction in pathogenes.

Molecular determinants for ATP-binding in proteins: a data mining and quantum chemical analysis.

Adenosine 5'-triphosphate (ATP) plays an essential role in all forms of life. Molecular recognition of ATP in proteins is a subject of great importance for understanding enzymatic mechanism and for drug design. We have carried out a large-scale data mining of the Protein Data Bank (PDB) to analyze molecular determinants for recognition of the adenine moiety of ATP by proteins. Non-bonded intermolecular interactions (hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) between adenine base and surrounding residues in its binding pockets are systematically analyzed for 68 non-redundant, high-resolution crystal structures of adenylate-binding proteins. In addition to confirming the importance of the widely known hydrogen bonding, we found out that cation-pi interactions between adenine base and positively charged residues (Lys and Arg) and pi-pi stacking interactions between adenine base and surrounding aromatic residues (Phe, Tyr, Trp) are also crucial for adenine binding in proteins. On average, there exist 2.7 hydrogen bonding interactions, 1.0 pi-pi stacking interactions, and 0.8 cation-pi interactions in each adenylate-binding protein complex. Furthermore, a high-level quantum chemical analysis was performed to analyze contributions of each of the three forms of intermolecular interactions (i.e. hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) to the overall binding force of the adenine moiety of ATP in proteins. Intermolecular interaction energies for representative configurations of intermolecular complexes were analyzed using the supermolecular approach at the MP2/6-311 + G* level, which resulted in substantial interaction strengths for all the three forms of intermolecular interactions. This work represents a timely undertaking at a historical moment when a large number of X-ray crystallographic structures of proteins with bound ATP ligands have become available, and when high-level quantum chemical analysis of intermolecular interactions of large biomolecular systems becomes computationally feasible. The establishment of the molecular basis for recognition of the adenine moiety of ATP in proteins will directly impact molecular design of ATP-binding site targeted enzyme inhibitors such as kinase inhibitors.

Downstream antibody purification using aqueous two-phase extraction.

The extraction of antibodies using a polyethylene glycol (PEG)-citrate aqueous two-phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6-fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product-related impurities (aggregates and low molecular product fragments) from ∼40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale-up model for the use of counter-current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode.

Insight into the structural role of carotenoids in the photosystem I: a quantum chemical analysis.

The structural stabilization role of carotenoids in the formation of photosynthetic pigment-protein complexes is investigated theoretically. The pi-pi stacking and CH-pi interactions between beta-carotenes and their surrounding chlorophylls (and/or aromatic residues) in Photosystem I (PS1) from the cyanobacterium Synechococcus elongatus were studied by means of the supermolecular approach at the level of the second-order Møller-Plesset perturbation method. PS1 features a core integral antenna system consisting of 22 beta-carotenes intertwined with 90 chlorophyll molecules. The binding environments of all 22 beta-carotenes were systematically analyzed. For 21 out of the 22 cases, one or more chlorophyll molecules exist within van der Waals' contacts of the beta-carotene molecule. The calculated strengths of pi-pi stacking interactions between the conjugated core of beta-carotene and the aromatic tetrapyrrole rings of chlorophyll are substantial, ranging from -3.54 kcal/mol for the perpendicular-positioned BCR4004...CHL1217 pair to -16.01 kcal/mol for the parallel-oriented BCR4007...CHL1122 pair. A strong dependence of the pi-pi stacking interaction energies on the intermolecular configurations of the two interacting pi-planes is observed. The parallel-oriented beta-carotene and chlorophyll pair is energetically much more stable than the perpendicular-positioned pair. The larger the extent of pi-pi overlapping, the stronger the interaction strength. In many cases, the beta-ring ends of beta-carotene molecules are found to interact with the tetrapyrrole rings of chlorophyll via CH-pi interactions. For the latter interactions, the calculated interaction strengths vary from -7.03 to -11.03 kcal/mol, depending on the intermolecular configuration. This work leads to the conclusion that pi-pi stacking and CH-pi interactions between beta-carotene and their surrounding chlorophylls and aromatic residues play an essential role in binding beta-carotenes in PS1 from S. elongatus. Consequently, the molecular basis of the structural stabilization function of carotenoids in formation of the photosynthetic pigment-protein complexes is established.

Multiple intermolecular interaction modes of positively charged residues with adenine in ATP-binding proteins.

Adenosine 5'-triphosphate (ATP) plays an essential role in all forms of life. Molecular recognition of ATP in ATP-binding proteins is a subject of great importance for understanding enzymatic mechanisms and for drug design. We have carried out a large-scale data mining of the Protein Data Bank (PDB) to analyze molecular determinants for recognition of ATP, in particular, the adenine base, by ATP-binding proteins. A novel distribution pattern of charged residues around the adenine base was discovered: lysine residues tend to occupy the major groove N7 side of the adenine base, and the arginine residues situate preferentially above or below the adenine bases. Such an arrangement is advantageous because it facilitates multiple modes of intermolecular interactions, that is, cation-pi interactions and a hydrogen bond between lysine and adenine, and cation-pi and pi-pi stacking interactions between arginine and adenine. For the two representative Lys... Adenine and Arg... Adenine interactions, intermolecular interaction energies were subsequently analyzed by means of the supermolecular approach at the MP2 level with solvation free energy correction using the SM5.42R model of Cramer and Truhlar, which gave rise to significant interaction strengths.