No abstract

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.

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.

Carotenoids play an important role of light harvesting, photoprotection and structural stabilization in the photosynthetic organisms. Despite their functional importance, the molecular basis for binding of carotenoids in the photosynthetic pigment-protein complexes is poorly understood. On the basis of a recent discovery that carotenoids are surrounded either by aromatic residues or by chlorophylls in all known crystal structures of the photosynthetic pigment-protein complexes (J. Am. Chem. Soc. 2002, 124, 8445), it is hypothesized that π-π stacking interactions are the molecular forces that bind carotenoids in the photosynthetic pigmentprotein complexes. In this article, the π-π stacking interactions between the carotenoid molecule peridinins and their surrounding aromatic groups (aromatic residues and chlorophyll-a) in the peridine-chlorophyllprotein complex of Amphidinium carterae are characterized by means of the supermolecular approach with the second-order Møller-Plesset perturbation method (MP2). The modified 6-31G*(0.25) basis set with diffuse d-polarization by Hobza et al. is adopted here. A representative peridinin chlorophyll pair (PID624‚‚‚Chl602) is chosen to study the structural stabilization role of peridinin, and three unique peridinin and aromatic residue pairs (PID623‚‚‚TYR270, PID624‚‚‚PHE301, and PID624‚‚‚PHE304) are chosen to study the configurational (orientation and distance) dependence of π-π stacking interactions between peridinins and their interacting partners. The MP2/6-31G*(0.25) calculations yielded a favorable π-π stacking interaction energy of -11.52 kcal/mol between the large conjugated tetrapyrrol π-system of chlorophyll Chl602 and the long conjugated π-electrons of peridinin PID624. For the parallelly oriented PID623‚‚‚TYR270 pair, the MP2/6-31G*(0.25) calculations give rise to a stabilization energy of -7.25 kcal/mol. For the perpendicularly oriented pairs, the calculated MP2/6-31G*(0.25) π-π stacking energies are -3.77 and -1.71 kcal/mol for PID624‚‚‚F301 and PID624‚‚‚F304, respectively. It is thus concluded that π-π stacking interactions between peridinins and the nearby aromatic groups play a substantial role in binding peridinins in the peridine-chlorophyll-protein complex of A. carterae. Consequently, the molecular basis of the structural stabilization function of carotenoids in forming the pigment-protein complexes is unraveled.

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.