[Regulatory effect of the pannexin1 channel on invasion and migration of testicular cancer Tcam-2 cells and its possible mechanism].

OBJECTIVE: To investigate the role of the pannexin-1 (Panx1) protein in the invasion and migration of testicular cancer Tcam-2 cells and its possible action mechanism. METHODS: Tcam-2 cells were treated with carbenoxolone (CBX) at 100 µmol/L and probenecid (PBN) 200 µmol/L. Then the intercellular fluorescence transmission was assessed by real-time fluorescence assay, the extracellular ATP concentration measured by chemi-luminescence immunoassay, the invasive and migratory abilities of the Tcam-2 cells detected by Transwell assay, and the expressions of the proteins Panx1, p-ERK1/2, ERK1/2, vimentin, MMP-9 and E-cadherin in the TM3 Leydig cells and testicular cancer Tcam-2 cells determined by Western blot. RESULTS: Western blot showed that the expression of the Panx1 protein was significantly higher in the testicular cancer Tcam-2 cells than in the TM3 Leydig cells (2.79 ± 0.17 vs 1.00 ± 0.06, P<0.05). The rates of intercellular fluorescence transmission in the Tcam-2 cells treated with CBX and PBN were markedly decreased as compared with the blank control group (ï¼»61.54 ± 3.30ï¼½% and ï¼»68.06 ± 4.03ï¼½% vs ï¼»99.50 ± 3.12ï¼½%, P<0.01), and so were the extracellular ATP concentrations (ï¼»57.06 ± 5.80ï¼½% and ï¼»56.42 ± 7.70ï¼½% vs ï¼»110 ± 8.16ï¼½%, P<0.01). The numbers of migrated Tcam-2 cells in the CBX and PBN groups were significantly reduced in comparison with that in the control (11.5 ± 1.11 and 8.25 ± 1.23 vs 331.00 ± 30.80, P<0.05), and so were those of the invaded ones (11.75 ± 3.77 and 11.5 ± 3.5 vs 89.00 ± 13.09, P<0.01). CBX and PBN significantly down-regulated the expression of p-ERK1/2 as compared with that in the blank control group (0.538 ± 0.05 and 0.476 ± 0.02 vs 0.98 ± 0.03, P<0.05), as well as those of vimentin (0.541 ± 0.09 and 0.705 ± 0.07, P<0.01) and MMP-9 (0.439 ± 0.08 and 0.557 ± 0.065, P<0.01) but up-regulated that of E-cadherin (3.896 ± 0.06 and 3.551 ± 0.04, P<0.01). CONCLUSIONS: The Panx1 protein is highly expressed in testicular cancer Tcam-2 cells. CBX and PBN can inhibit the function of the panneixn1 channel and reduce the invasive and migratory abilities of the Tcam-2 cells, which is associated with the decreased expression of the p-ERK1/2 protein.

Molecular insight into conformational transmission of human P-glycoprotein.

P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

Exploring the inter-molecular interactions in amyloid-ß protofibril with molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area free energy calculations.

Aggregation of amyloid-ß (Aß) peptides correlates with the pathology of Alzheimer's disease. However, the inter-molecular interactions between Aß protofibril remain elusive. Herein, molecular mechanics Poisson-Boltzmann surface area analysis based on all-atom molecular dynamics simulations was performed to study the inter-molecular interactions in Aß(17-42) protofibril. It is found that the nonpolar interactions are the important forces to stabilize the Aß(17-42) protofibril, while electrostatic interactions play a minor role. Through free energy decomposition, 18 residues of the Aß(17-42) are identified to provide interaction energy lower than -2.5 kcal/mol. The nonpolar interactions are mainly provided by the main chain of the peptide and the side chains of nine hydrophobic residues (Leu17, Phe19, Phe20, Leu32, Leu34, Met35, Val36, Val40, and Ile41). However, the electrostatic interactions are mainly supplied by the main chains of six hydrophobic residues (Phe19, Phe20, Val24, Met35, Val36, and Val40) and the side chains of the charged residues (Glu22, Asp23, and Lys28). In the electrostatic interactions, the overwhelming majority of hydrogen bonds involve the main chains of Aß as well as the guanidinium group of the charged side chain of Lys28. The work has thus elucidated the molecular mechanism of the inter-molecular interactions between Aß monomers in Aß(17-42) protofibril, and the findings are considered critical for exploring effective agents for the inhibition of Aß aggregation.

Molecular basis for polyol-induced protein stability revealed by molecular dynamics simulations.

Molecular dynamics simulations of chymotrypsin inhibitor 2 in different polyols (glycerol, xylitol, sorbitol, trehalose, and sucrose) at 363 K were performed to probe the molecular basis of the stabilizing effect, and the data in water, ethanol, and glycol were compared. It is found that protein protection by polyols is positively correlated with both the molecular volume and the fractional polar surface area, and the former contributes more significantly to the protein's stability. Polyol molecules have only a few direct hydrogen bonds with the protein, and the number of hydrogen bonds between a polyol and the protein is similar for different polyols. Thus, it is concluded that the direct interactions contribute little to the stabilizing effect. It is clarified that the preferential exclusion of the polyols is the origin of their protective effects, and it increases with increasing polyol size. Namely, there is preferential hydration on the protein surface (2 A), and polyol molecules cluster around the protein at a distance of about 4 A. The preferential exclusion of polyols leads to indirect interactions that prevent the protein from thermal unfolding. The water structure becomes more ordered with increasing the polyol size. So, the entropy of water in the first hydration shell decreases, and a larger extent of decrease is observed with increasing polyol size, leading to larger transfer free energy. The findings suggest that polyols protect the protein from thermal unfolding via indirect interactions. The work has thus elucidated the molecular mechanism of structural stability of the protein in polyol solutions.

Molecular insight into the inhibition effect of trehalose on the nucleation and elongation of amyloid beta-peptide oligomers.

Soluble amyloid oligomers are a cytotoxic species in Alzheimer's disease, and the recent discovery that trehalose can prohibit aggregation of amyloid beta-peptide (Abeta) has received great attention. However, its inhibition mechanism remains unclear. In order to investigate the molecular mechanism of the inhibition effect, molecular dynamics simulations of Abeta(16-22) and Abeta(40) peptides at different trehalose concentrations (0-0.18 mol/L) are performed using an all-atom model. The simulations confirmed that Abeta(16-22) aggregation is prevented by trehalose in a dose-dependent manner, and it is found that the preferential exclusion effect of trehalose is the origin of its inhibition effects. Namely, there is preferential hydration on the peptide surface (3 A), and trehalose molecules cluster around the peptides at a distance of 4-5 A. At high trehalose concentrations, the preferential exclusion of trehalose leads to three sequential effects that prevent the nucleation and elongation of Abeta(16-22) oligomers. First, the secondary structures of Abeta(16-22) monomers are stabilized in the turn, bend, or coil, so the beta-sheet-rich structure that is prone to forming peptide oligomers is prevented. Second, the thin hydration layer and trehalose clusters can weaken hydrophobic interactions that lead to Abeta(16-22) aggregation. Third, more direct and indirect H-bonds form between trehalose and Abeta(16-22), which suppress the interpeptide hydrogen bonding. Analyses of the simulation data for a single Abeta(40) peptide indicate that trehalose can inhibit the nucleation and elongation of Abeta(40) by a similar mechanism with that on Abeta(16-22) oligomerization. The work has thus elucidated the molecular mechanism of trehalose on the inhibition of Abeta oligomeric aggregation.

Rational design of peptide ligand for affinity chromatography of tissue-type plasminogen activator by the combination of docking and molecular dynamics simulations.

Combined docking and molecular dynamics (MD) simulations are carried out for the rational design of affinity peptide ligand of tissue-type plasminogen activator (t-PA). Ten amino acids that have high affinity to three different regions of t-PA are identified by the amino acids location method on the basis of candidate pocket structure of t-PA. Then, 14 tetrapeptides are built and docked into the candidate pocket of t-PA. The absolute value of the D(score) calculated from the docking simulation is used to assess the affinity of a peptide for t-PA. Consequently, six tetrapeptides that have high D(score) values are selected and linked to a spacer arm of [NH(CH(2))(6)NH(2)] that is present on EAH Sepharose gel. The linked compounds are further evaluated by docking into the candidate pocket of t-PA. As a result, the tetrapeptide QDES with the highest D(score) value is selected. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions and hydrogen bonds (H-bonds) contribute to the affinity interactions between the tetrapeptide and t-PA. MD simulations indicate that QDES-t-PA complex keeps stable, and the distances between the carboxyl groups of Asp189, Gln192 and Asp194 and the charged amino group of glutamine change little. Moreover, all the nine H-bonds found in the docking simulation are confirmed by the MD simulations. It is also found that three water molecules act as bridges between the ligand and the protein pocket by hydrogen bonding. Finally, high binding affinity and specificity of the peptide ligand are confirmed by the purification of t-PA from crude porcine heart extract using the immobilized-ligand column for affinity chromatography.

Rational design of affinity peptide ligand by flexible docking simulation.

Rational design of affinity peptide ligands of proteins by flexible docking simulation is performed using the SYBYL program package. This approach involves the use of experimental data to verify a scoring function that can be used to assess the affinity of a peptide for its target protein. The enzyme-linked immunosorbent assay (ELISA) data of several peptides displayed on phage surfaces for insulin and lysozyme, respectively, reported in literature are used for the purpose. It is found that the absolute values of the Dscore calculated from the docking correspond well to the ELISA data that relate to the affinity between the peptides and the target molecule. So, the Dscore function is used to assess the affinity of docked peptides in a pentapeptide library designed on the basis of protein (alpha-amylase) structure. As a result, a pentapeptide with a high Dscore value is selected and a hexapeptide (FHENWS) is built by linking serine to its C-terminal to lengthen the peptide. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions (including hydrogen bonds) and Van der Waals forces contribute to the affinity of the hexapeptide for alpha-amylase. Chromatographic experiments with the immobilized peptide have given further evidence for this observation. Adsorption isotherm described by the Langmuir equation indicates that the apparent binding constant of alpha-amylase to the immobilized hexapeptide was 2.5x10(5)L/mol. Finally, high affinity and specificity of the affinity adsorbent is exemplified by the purification of alpha-amylase from crude fermentation broth of Bacillus subtilis.

Design of LVFFARK and LVFFARK-functionalized nanoparticles for inhibiting amyloid ß-protein fibrillation and cytotoxicity.

Aggregation of amyloid ß-protein (Aß) into amyloid oligomers and fibrils is pathologically linked to Alzheimer's disease (AD). Hence, the inhibition of Aß aggregation is essential for the prevention and treatment of AD, but the development of potent agents capable of inhibiting Aß fibrillogenesis has posed significant challenges. Herein, we designed Ac-LVFFARK-NH2 (LK7) by incorporating two positively charged residues, R and K, into the central hydrophobic fragment of Aß17-21 (LVFFA) and examined its inhibitory effect on Aß42 aggregation and cytotoxicity by extensive physical, biophysical, and biological analyses. LK7 was observed to inhibit Aß42 fibrillogenesis in a dose-dependent manner, but its strong self-assembly characteristic also resulted in high cytotoxicity. In order to prevent the cytotoxicity that resulted from the self-assembly of LK7, the peptide was then conjugated to the surface of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to fabricate a nanosized inhibitor, LK7@PLGA-NPs. It was found that LK7@PLGA-NPs had little cytotoxicity because the self-assembly of the LK7 conjugated on the NPs was completely inhibited. Moreover, the NPs-based inhibitor showed remarkable inhibitory capability against Aß42 aggregation and significantly alleviated its cytotoxicity at a low LK7@PLGA-NPs concentration of 20 µg/mL. At the same peptide concentration, free LK7 showed little inhibitory effect. It is considered that several synergetic effects contributed to the strong inhibitory ability of LK7@PLGA-NPs, including the enhanced interactions between Aß42 and LK7@PLGA-NPs brought on by inhibiting LK7 self-assembly, restricting conformational changes of Aß42, and thus redirecting Aß42 aggregation into unstructured, off-pathway aggregates. The working mechanisms of the inhibitory effects of LK7 and LK7@PLGA-NPs on Aß42 aggregation were proposed based on experimental observations. This work provides new insights into the design and development of potent NPs-based inhibitors against Aß aggregation and cytotoxicity.

Brazilin inhibits amyloid ß-protein fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity.

Soluble amyloid ß-protein (Aß) oligomers, the main neurotoxic species, are predominantly formed from monomers through a fibril-catalyzed secondary nucleation. Herein, we virtually screened an in-house library of natural compounds and discovered brazilin as a dual functional compound in both Aß42 fibrillogenesis inhibition and mature fibril remodeling, leading to significant reduction in Aß42 cytotoxicity. The potent inhibitory effect of brazilin was proven by an IC50 of 1.5 ± 0.3 µM, which was smaller than that of (-)-epigallocatechin gallate in Phase III clinical trials and about one order of magnitude smaller than those of curcumin and resveratrol. Most importantly, it was found that brazilin redirected Aß42 monomers and its mature fibrils into unstructured Aß aggregates with some ß-sheet structures, which could prevent both the primary nucleation and the fibril-catalyzed secondary nucleation. Molecular simulations demonstrated that brazilin inhibited Aß42 fibrillogenesis by directly binding to Aß42 species via hydrophobic interactions and hydrogen bonding and remodeled mature fibrils by disrupting the intermolecular salt bridge Asp23-Lys28 via hydrogen bonding. Both experimental and computational studies revealed a different working mechanism of brazilin from that of known inhibitors. These findings indicate that brazilin is of great potential as a neuroprotective and therapeutic agent for Alzheimer's disease.

Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations.

Molecular basis for the dissociation dynamics of protein A-immunoglobulin G1 complex.

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.

Molecular insight into the counteraction of trehalose on urea-induced protein denaturation using molecular dynamics simulation.

Considerable experimental evidence indicates that trehalose can counteract the denaturing effects of urea on proteins. However, its molecular mechanism remains unknown due to the limitations of current experimental techniques. Herein, molecular dynamics simulations were performed to investigate the counteracting effects of trehalose against urea-induced denaturation of chymotrypsin inhibitor 2. The simulations indicate that the protein unfolds in 8 mol/L urea, but at the same condition the protein retains its native structure in the ternary solution of 8 mol/L urea and 1 mol/L trehalose. It is confirmed that the preferential exclusion of trehalose from the protein surface is the origin of its counteracting effects. It is found that trehalose binds urea via hydrogen bonds, so urea molecules are also expelled from the protein surface along with the preferential exclusion of trehalose. The exclusion of urea from the protein surface leads to the alleviation of the Lennard-Jones interactions between urea and the hydrophobic side chains of the protein in the ternary solution. In contrast, the electrostatic interactions between urea and the protein change little in the presence of trehalose because the decrease in the electrostatic interactions between urea and the protein backbone is canceled by the increase in the electrostatic interactions between urea and the charged side chains of the protein. The results have provided molecular explanations for the counteraction of urea-induced protein denaturation by trehalose.

Molecular mechanism of the effects of salt and pH on the affinity between protein A and human immunoglobulin G1 revealed by molecular simulations.

Protein A from the bacterium Staphylococcus aureus (SpA) has been widely used as an affinity ligand for purification of immunoglobulin G (IgG). The affinity between SpA and IgG is affected differently by salt and pH, but their molecular mechanisms still remain unclear. In this work, molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area analysis were performed to investigate the salt (NaCl) and pH effects on the affinity between SpA and human IgG1 (hIgG1). It is found that salt and pH affect the interactions of the hot spots of SpA by different mechanisms. In the salt solution, the compensations between helices I and II of SpA as well as between the nonpolar and electrostatic energies make the binding free energy independent of salt concentration. At pH 3.0, the unfavorable electrostatic interactions increase greatly and become the driving force for dissociation of the SpA-hIgG1 complex. They mainly come from the strong electrostatic repulsions between positively charged residues (H137, R146, and K154) of SpA and the positively charged residues of hIgG1. It is considered to be the molecular basis for hIgG1 elution from SpA-based affinity adsorbents at pH 3.0. The dissociation mechanism is then used to refine the binding model of SpA to hIgG1. The model is expected to help design high-affinity peptide ligands of IgG.

Molecular mechanism of the affinity interactions between protein A and human immunoglobulin G1 revealed by molecular simulations.

Protein A (SpA) affinity chromatography has been widely used for the purification of immunoglobulin G (IgG). However, the molecular mechanism of the affinity between IgG and SpA remains unclear. In this work, molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area analysis were performed to investigate the molecular mechanism of the affinity interactions. It is found that hydrophobic interaction contributes more than 80% to the binding free energy, while electrostatic interaction plays a minor role (<20%). Through free energy decomposition and pair interaction analysis, the hot spots of the SpA-hIgG1 complex are identified. For hIgG1, the hot spots include the residues of I253, H310, Q311, D315, K317, E430, and N434. For SpA, residues F132, Y133, H137, E143, R146, and K154 contribute significantly. Furthermore, helix I of SpA binds Fc through hydrophobic interaction, while helix II mainly provides electrostatic interaction that determines the binding selectivity to different Igs. Finally, the binding motif of SpA is constructed, which would help design novel high-affinity ligands of IgG.

Molecular insight into conformational transition of amyloid ß-peptide 42 inhibited by (-)-epigallocatechin-3-gallate probed by molecular simulations.

Considerable experimental evidence indicates that (-)-epigallocatechin-3-gallate (EGCG) inhibits the fibrillogenesis of Aß(42) and alleviates its associated cytotoxicity. However, the molecular mechanism of the inhibition effect of EGCG on the conformational transition of Aß(42) remains unclear due to the limitations of current experimental techniques. In this work, molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis were coupled to better understand the issue. It was found that the direct interactions between EGCG and the peptide are the origin of its inhibition effects. Specifically, EGCG molecules expel water from the surface of the Aß(42), cluster with each other, and interact directly with the peptide. The results of free energy decomposition calculated by MM-PBSA indicate that the nonpolar term contributes more than 71% to the binding free energy of the EGCG-Aß(42) complex, while polar interactions (i.e., hydrogen bonding) play a minor role. It was identified that there are 12 important residues of Aß(42) that strongly interact with EGCG (Phe4, Arg5, Phe19, Phe20, Glu22, Lys28, Gly29, Leu34-Gly37, and Ile41), while nonpolar interactions are mainly provided by the side chains of some hydrophobic residues (Phe, Met and Ile) and the main chains of some nonhydrophobic residues (Lys28 and Gly29). On the contrary, polar interactions are mainly formed by the main chain of Aß(42), of which the main chains of Gly29 and Gly37 contribute greatly. The work has thus elucidated the molecular mechanism of the inhibition effect of EGCG on the conformational transition of Aß(42), and the findings are considered critical for exploring more effective agents for the inhibition of Aß(42) fibrillogenesis.

Thermodynamic analysis of the molecular interactions between amyloid beta-peptide 42 and (-)-epigallocatechin-3-gallate.

One of the key factors of Alzheimer's disease (AD) is the conversion of amyloid beta-peptide (Abeta) from its soluble random coil form into various aggregated forms. (-)-Epigallocatechin-3-gallate (EGCG) has been proved effective in preventing the aggregation of Abeta, but the thermodynamic mechanisms are still unclear. In this work, isothermal titration calorimetry (ITC) was utilized to study the interactions between Abeta42 and EGCG at different temperatures, salt concentrations, pH values, and EGCG and Abeta42 concentrations. Molecular dynamics (MD) simulations were performed to study the hydrogen bonding between Abeta42 and EGCG. The results indicate that the binding stoichiometry N is linearly related to the EGCG/Abeta42 ratio. Hydrophobic interaction and hydrogen bonding are both substantial in the binding process, but the extent of their contributions changes with experimental conditions. Namely, the predominant interaction gradually shifts from a hydrogen bonding to a hydrophobic interaction with the increase of the EGCG/Abeta42 ratio, resulting in a transition of the binding from enthalpy-driven to entropy-driven. This experimental observation is validated by the MD simulations. The binding of EGCG to Abeta42 can be promoted by increasing temperature and salt concentration and changing pH away from Abeta42's pI. The findings have provided new insight into the molecular interactions between Abeta42 and EGCG from a thermodynamic perspective and are expected to facilitate the research on the inhibition of Abeta42 aggregation.

Approaches to high-performance preparative chromatography of proteins.

Preparative liquid chromatography is widely used for the purification of chemical and biological substances. Different from high-performance liquid chromatography for the analysis of many different components at minimized sample loading, high-performance preparative chromatography is of much larger scale and should be of high resolution and high capacity at high operation speed and low to moderate pressure drop. There are various approaches to this end. For biochemical engineers, the traditional way is to model and optimize a purification process to make it exert its maximum capability. For high-performance separations, however, we need to improve chromatographic technology itself. We herein discuss four approaches in this review, mainly based on the recent studies in our group. The first is the development of high-performance matrices, because packing material is the central component of chromatography. Progress in the fabrication of superporous materials in both beaded and monolithic forms are reviewed. The second topic is the discovery and design of affinity ligands for proteins. In most chromatographic methods, proteins are separated based on their interactions with the ligands attached to the surface of porous media. A target-specific ligand can offer selective purification of desired proteins. Third, electrochromatography is discussed. An electric field applied to a chromatographic column can induce additional separation mechanisms besides chromatography, and result in electrokinetic transport of protein molecules and/or the fluid inside pores, thus leading to high-performance separations. Finally, expanded-bed adsorption is described for process integration to reduce separation steps and process time.

Molecular mechanism for the effects of trehalose on beta-hairpin folding revealed by molecular dynamics simulation.

Recent work has shown that trehalose can facilitate and inhibit protein folding, but little is known about the molecular basis of these effects. Molecular-level insights into how the osmolyte affects protein folding are of significance for the rational design of small molecular additives for enhancing or hindering the folding of proteins. To investigate the molecular mechanisms of the facilitation and inhibition effects of trehalose on protein folding, molecular dynamics (MD) simulation of a beta-hairpin peptide (Trp-Arg-Tyr-Tyr-Glu-Ser-Ser-Leu-Glu-Pro-Glu-Pro-Asp) in different trehalose concentrations (0-0.26 mol/L) is performed using an all-atom model. It is found that at a proper trehalose concentration (0.065 mol/L), the peptide folds faster than that in water, but it cannot fold to the beta-hairpin at higher trehalose concentrations. Free energy landscape analysis indicates the presence of three intermediate states in both pure water and in 0.065 mol/L trehalose, but the potential energy barriers in the folding pathway decrease greatly in 0.065 mol/L trehalose, so the peptide folding is facilitated. Moreover, at this trehalose concentration, there is a favorable balance between the peptide backbone hydrogen bonds (H-bonds) and the peptide-trehalose H-bonds, leading to the stabilization of the folded peptide. At higher trehalose concentrations, however, trehalose molecules cluster in the peptide region and interact with the peptide via many H-bonds that prevent the peptide from folding to its native structure. The energy landscape analysis indicates that the potential energy barriers increase so greatly that the peptide cannot overcome it, getting trapped in a local free energy basin. The work reported herein has elucidated the molecular mechanism of the peptide folding in the presence of trehalose.