A Physical Security Based Mailing System for Self Destructive Databases

Abstract: Persona

Aspects of Weak Interactions between Folate and Glycine Betaine

Folate, or vitamin B₉, is an important compound in one-carbon metabolism. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equilibrium toward the free ligand and protein species. This study uses vapor-pressure osmometry to explore the interaction of folate with the model osmolyte, glycine betaine. This method yields a preferential interaction potential (μ₂₃/<i>RT</i> value). This value is concentration-dependent as folate dimerizes. The μ₂₃/<i>RT</i> value also tracks the deprotonation of folate’s N3–O4 keto–enol group, yielding a p<i>K</i>_a of 8.1. To determine which folate atoms interact most strongly with betaine, the interaction of heterocyclic aromatic compounds (as well as other small molecules) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the μ₂₃/<i>RT</i> values into α values for atom types was achieved. This allows prediction of μ₂₃/<i>RT</i> values for larger molecules such as folate. Molecular dynamics simulations of folate show a variety of structures from extended to L-shaped. These conformers possess μ₂₃/<i>RT</i> values from −0.18 to 0.09 <i>m</i>^–1, where a negative value indicates a preference for solvation by betaine and a positive value indicates a preference for water. This range of values is consistent with values observed in osmometry and solubility experiments. As the average predicted folate μ₂₃/<i>RT</i> value is near zero, this indicates folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water, while the aromatic rings prefer betaine. In general, the more protonated species in our small molecule survey interact better with betaine as they provide a source of hydrogens (betaine is not a hydrogen bond donor). Upon deprotonation of the small molecule, the preference swings toward water interaction because of its hydrogen bond donating capacities

Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases

A weak association between osmolytes and dihydrofolate (DHF) decreases the affinity of the substrate for the <i>Escherichia coli</i> chromosomal and R67 plasmid dihydrofolate reductase (DHFR) enzymes. To test whether the osmolyte–DHF association also interferes with binding of DHF to FolM, an <i>E. coli</i> enzyme that possesses weak DHFR activity, ligand binding was monitored in the presence of osmolytes. The affinity of FolM for DHF, measured by <i>k</i>_cat/<i>K</i>_m(DHF), was decreased by the addition of an osmolyte. Additionally, binding of the antifolate drug, methotrexate, to FolM was weakened by the addition of an osmolyte. The changes in ligand binding with water activity were unique for each osmolyte, indicating preferential interaction between the osmolyte and folate and its derivatives; however, additional evidence provided support for further interactions between FolM and osmolytes. Binding of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) cofactor to FolM was monitored by isothermal titration calorimetry as a control for protein–osmolyte association. In the presence of betaine (proposed to be the osmolyte most excluded from protein surfaces), the NADPH <i>K</i>_d decreased, consistent with dehydration effects. However, other osmolytes did not tighten binding to the cofactor. Rather, dimethyl sulfoxide (DMSO) had no effect on the NADPH <i>K</i>_d, while ethylene glycol and polyethylene glycol 400 weakened cofactor binding. Differential scanning calorimetry of FolM in the presence of osmolytes showed that both DMSO and ethylene glycol decreased the stability of FolM, while betaine increased the stability of the protein. These results suggest that some osmolytes can destabilize FolM by preferentially interacting with the protein. Further, these weak attractions can impede ligand binding. These various contributions have to be considered when interpreting osmotic pressure results

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N‑Termini

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of <i>Escherichia coli</i> DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (<i>R</i>_g) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the <i>R</i>_g increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated

Aspects of Weak Interactions between Folate and Glycine Betaine

Folate, or vitamin B₉, is an important compound in one-carbon metabolism. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equilibrium toward the free ligand and protein species. This study uses vapor-pressure osmometry to explore the interaction of folate with the model osmolyte, glycine betaine. This method yields a preferential interaction potential (μ₂₃/<i>RT</i> value). This value is concentration-dependent as folate dimerizes. The μ₂₃/<i>RT</i> value also tracks the deprotonation of folate’s N3–O4 keto–enol group, yielding a p<i>K</i>_a of 8.1. To determine which folate atoms interact most strongly with betaine, the interaction of heterocyclic aromatic compounds (as well as other small molecules) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the μ₂₃/<i>RT</i> values into α values for atom types was achieved. This allows prediction of μ₂₃/<i>RT</i> values for larger molecules such as folate. Molecular dynamics simulations of folate show a variety of structures from extended to L-shaped. These conformers possess μ₂₃/<i>RT</i> values from −0.18 to 0.09 <i>m</i>^–1, where a negative value indicates a preference for solvation by betaine and a positive value indicates a preference for water. This range of values is consistent with values observed in osmometry and solubility experiments. As the average predicted folate μ₂₃/<i>RT</i> value is near zero, this indicates folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water, while the aromatic rings prefer betaine. In general, the more protonated species in our small molecule survey interact better with betaine as they provide a source of hydrogens (betaine is not a hydrogen bond donor). Upon deprotonation of the small molecule, the preference swings toward water interaction because of its hydrogen bond donating capacities

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N‑Termini

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of <i>Escherichia coli</i> DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (<i>R</i>_g) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the <i>R</i>_g increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated