Joseph C.P Koo

Glutamate Transporter Coupling to Na,K-ATPase

Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The α1–α3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using [3H]d-aspartate, [3H]l-glutamate, and rubidium-86 uptake into synaptosomes and cultured astrocytes. In synaptosomes, ouabain produced a dose-dependent inhibition of glutamate transporter and Na,K-ATPase activities, whereas in astrocytes, ouabain showed a bimodal effect whereby glutamate transporter activity was stimulated at 1 μm ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [3H]d-aspartate uptake indicated the selective involvement of Src kinases, which are probably a component of the Na,K-ATPase/glutamate transporter complex. These findings demonstrate that glutamate transporters and Na,K-ATPases are part of the same macromolecular complexes and operate as a functional unit to regulate glutamatergic neurotransmission.

Generation and analysis of the conformational potential energy surfaces of N-acetyl-N-methyl-L-alanine-N'-methylamide. An exploratory ab initio study

Abstract N-methylation is a naturally occurring modification in small peptides, e.g. antibiotics that can effect the conformational preferences of the molecule as well as the ease of trans to cis isomerization of the involved peptide bond. In the present exploratory study we have calculated the potential energy surface of both N -acetyl- l -alanine- N ′-methylamide and N -acetyl- N -methyl- l -alanine- N ′-methylamide at the RHF/3-21G level of theory with a cis – trans or with a trans – trans peptide conformation. With respect to the non-methylated model system our results indicate that N-methylation reduces the number of observable backbone conformers in both amide configurations. The effect of methylation on the ease of trans to cis isomerization was assessed by calculating the energetics of the corresponding transition structures. An increase in the activation energies of the trans to cis isomerization of the relevant peptide bond was observed for the N-methylated moiety.

Comprehensive conformational analysis of N-acetyl-l-isoleucine-N-methylamide: an ab initio study

Abstract A conformational study on N -acetyl- l -isoleucine- N -methylamide was carried out using ab initio (RHF/3-21G and RHF/6-31G(d)) calculations. All backbone and side-chain conformations in this compound were explored. N -acethyl- l -isoleucine- N -methylamide displayed a significant molecular flexibility. Also a different conformational behaviour was obtained for this amino acid residue in comparison with other amino acids possessing shorter apolar side-chains. These results can be attributed, at least in part, to the side-chain/backbone interactions, which are stabilizing the low-energy conformations in this molecule.

How reliable could economic Hartree–Fock computations be in studying large, folded peptides? A comparative HF and DFT case study on N- and C-protected aspartic acid

Abstract In this study, potential energy hypersurfaces have been generated and analyzed for each of the nine possible backbone (BB) conformations for both the endo and exo forms of N-acetyl- l -aspartic acid N′-methylamide. Ab initio calculations were carried out at RHF/3-21G, RHF/6-31G(d), and B3LYP/6-31G(d) levels for all backbone conformations. The relative energies, as well as stabilization energies exerted by the sidechain (SC) on the backbone, were calculated for all stable conformers. All sidechain–sidechain (HO⋯OC), backbone–backbone (N–H⋯OC), and sidechain–backbone (N–H⋯OC; N–H⋯OH) hydrogen bond interactions were analyzed. The appearance of the traditionally absent αL and eL conformers may be recognized as special geometric orientation which the aspartyl residue manifests during peptide folding or ligand docking in a receptor that contains aspartic acids in its ligand recognition sites. At all three levels of theory, there exists a trend between the hydrogen bond distance and ring size. In addition, strikingly high correlations between the torsional angles (R2=0.9937 for RHF/6-31G(d) versus RHF/3-21G; R2=0.9967 for B3LYP/6-31G(d) versus RHF/6-31G(d); R2=0.9914 for B3LYP/6-31G(d) versus RHF/3-21G) and between the ΔE values in kcal/mol (R2=0.9424 for RHF/6-31G(d) versus RHF/3-21G; R2=0.9108 for B3LYP/6-31G(d) versus RHF/6-31G(d); R2=0.9434 B3LYP/6-31G(d) versus RHF/3-21G) found at the different ab initio levels suggest that calculations carried out at the lower levels (i.e. at RHF/3-21G) are still significant.

Conformational dependence of the intrinsic acidity of the aspartic acid residue sidechain in N-acetyl-l-aspartic acid-N′-methylamide

Abstract The sidechain conformational potential energy hypersurfaces (PEHS) for the γL, βL, αL, and αD backbone conformations of N-acetyl- l -aspartate-N′-methylamide were generated. Of the 81 possible conformers initially expected for the aspartate residue, only seven were found after geometric optimizations at the B3LYP/6-31G(d) level of theory. No stable conformers could be located in the δL, eL, γD, δD, and eD backbone conformations. The ‘adiabatic’ deprotonation energies for the endo and exo forms of N-acetyl- l -aspartic acid-N′-methylamide were calculated by comparing their optimized relative energies against those found for the seven stable conformers of N-acetyl- l -aspartate-N′-methylamide. Sidechain conformational PEHSs were also generated for the estimation of ‘vertical’ deprotonation energies for both endo and exo forms of N-acetyl- l -aspartic acid-N′-methylamide. All backbone–sidechain (N–H⋯−O–C) and backbone–backbone (N–H⋯OC) hydrogen bond interactions were analyzed. A total of two backbone–backbone and four backbone–sidechain interactions were found for N-acetyl- l -aspartate-N′-methylamide. The deprotonated sidechain of N-acetyl- l -aspartate-N′-methylamide may allow the aspartyl residue to form strong hydrogen bond interactions (since it is negatively charged) which may be significant in such processes as protein–ligand recognition and ligand binding. As a primary example, the molecular geometry of the aspartyl residue may be important in peptide folding, such as that in the RGD tripeptide.