New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.

CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. In addition, the CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This paper provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM paper in 1983.

P arkinson's disease (PD) is the second most prevalent neurodegenerative disorder, but the etiology remains poorly understood (1, 2). Patients with PD suffer from rigidity, slowness of movement, tremor, and disturbances of balance (1, 2). PD is characterized by the progressive loss of dopamine-containing neurons in the substantia nigra pars compacta (3, 4) and the accumulation of Lewy bodies, proteinaceous intracytoplasmic accumulations of eosinophilic material that stain for ubiquitin (5). Novel insights into the molecular mechanisms of the pathogenesis of PD have come from the identification of genes associated with rare forms of familial PD (6). Mutations in ␣-synuclein (A53T and A30P) are linked to autosomal dominant PD (7,8). This led to the discovery that ␣-synuclein is a major component of Lewy bodies and suggests that derangements in ␣-synuclein could be a major cause or contributor to the pathogenesis of sporadic PD (9, 10). Consistent with this notion are observations that overexpression of ␣-synuclein in transgenic fruit flies and mice causes a Parkinsonian phenotype and replicates many of the pathological features of PD (11-13).Other autosomal dominant genes or loci linked to PD have been described and include a mutation (I93M) in ubiquitin carboxyl-terminal-hydrolase-L1 (14), a member of the ubiquitin C-terminal hydrolase family that hydrolyzes small C-terminal adducts of ubiquitin to generate ubiquitin monomers and is involved in facilitating the degradation and processing of proteins through the 26 S proteasome. Thus, derangements in ubiquitin processing may be linked to the pathogenesis of PD. Linkages to chromosome 2P and 4P have been described, as well as yet to be identified loci, but the genes await identification (15-17).Mutations in the Parkin gene are responsible for autosomal recessive PD (18). Several Parkin-associated pedigrees have been described with both deletions and point mutations, as well as compound heterozygosity causing autosomal recessive PD (19)(20)(21). Recent studies suggest that mutations in Parkin are the major cause of autosomal recessive familial PD (19); thus, understanding the function of Parkin and how mutations interfere with the function of Parkin may provide novel insights into the pathogenesis of PD. The function of the Parkin protein remains unknown. However, Parkin shows mild homology to ubiquitin at the N terminus and contains two ring-finger motifs and an in-between ring-finger (IBR) domain at the C terminus (22). Recently, a few proteins with ring-finger motifs similar to Parkin were shown to be involved in E2-dependent ubiquitination (23-26). Ubiquitination requires the ATP-dependent activation of ubiquitin by the ubiquitin-activating enzyme E1. Ubiquitin is transferred to an E2 ubiquitin-conjugating enzyme, which works in conjunction with an E3 ubiquitin-protein ligase to ubiquitinate substrate proteins (23,24). The existence of two ring-finger motifs and the N-terminal homology to ubiquitin suggests that Parkin may be involved in the ubiquitination pathw...

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.

Adipose differentiation related protein (ADRP) is a 50-kDa novel protein cloned from a mouse 1246 adipocyte cDNA library, rapidly induced during adipocyte differentiation. We have examined ADRP function, and we show here that ADRP facilitates fatty acid uptake in COS cells transfected with ADRP cDNA. We demonstrate that uptake of long chain fatty acids was significantly stimulated in a time-dependent fashion in ADRPexpressing COS-7 cells compared with empty vectortransfected control cells. Oleic acid uptake velocity increased significantly in a dose-dependent manner in ADRP-expressing COS-7 cells compared with control cells. The transport K m was 0.051 M, and V max was 57.97 pmol/10 5 cells/min in ADRP-expressing cells, and K m was 0.093 M and V max was 20.13 pmol/10 5 cells/min in control cells. The oleate uptake measured at 4°C was only 10% that at 37°C. ADRP also stimulated uptake of palmitate and arachidonate but had no effect on uptake of medium chain fatty acid such as octanoic acid and glucose. These data suggest that ADRP specifically enhances uptake of long chain fatty acids by increasing the initial rate of uptake and provide novel information about ADRP function as a saturable transport component for long chain fatty acids.Long chain non-esterified free fatty acids (FA) 1 and their derivatives have multiple functions as either essential components of membrane, efficient sources of energy, or important effective molecules that regulate metabolism and mediate gene expression (1, 2). Adipose tissue is the main source of lipids and fatty acids in the body where they play key roles in the regulation of energy balance in mammals. Proteins involved in FA and triglyceride synthesis and accumulation as well as utilization of exogenous lipid are induced during adipocyte differentiation (3, 4). Increase of enzymatic activities regulating lipogenesis and lipolysis is also ongoing at this stage. These multiple roles of FA suggest that careful regulation of cellular aspects of FA metabolism including cellular uptake in liver, fat, cardiac and skeletal muscles, and other organs is essential.The mechanism by which long chain free FA enter the cells is not completely understood. It has long been postulated that the movement of long chain FA across the cell membrane is invariably passive (5, 6). Studies (5, 6) have suggested that FA penetrate cardiac myocytes by a passive unregulated mechanism rather than by a specific facilitated process and that saturation of an intracellular metabolism step is the cause for apparent saturation of uptake in other studies. Others (7) have also shown that the entry of FA into hepatocytes reflected their passive partitioning into the lipid component of the cell membrane.

To identify possible relationships between the loss of calcium homeostasis in brain associated with aging and alterations in the function of key calcium regulatory proteins, we have purified calmodulin (CaM) from the brains of Fischer 344 rats of different ages and have assessed age-related alterations in (i) the secondary and tertiary structure of CaM and (ii) the ability of CaM to activate one of its target proteins, the plasma membrane (PM) Ca-ATPase. There is a progressive, age-dependent reduction in the ability of CaM to activate the PM-Ca-ATPase, which correlates with the oxidative modification of multiple methionines to their corresponding methionine sulfoxides. No other detectable age-related posttranslational modifications occur in the primary sequence of CaM, suggesting that the reduced ability of CaM to activate the PM-Ca-ATPase is the result of methionine oxidation. Corresponding age-related changes in the secondary and tertiary structure of CaM occur, resulting in alterations in the relative mobility of CaM on polyacrylamide gels, differences in the intrinsic fluorescence intensity and solvent accessibility of Tyr99 and Tyr138, and a reduction in the average alpha-helical content of CaM at 20 degreesC. Shifts in the calcium- and CaM-dependent activation of the PM-Ca-ATPase are observed for CaM isolated from senescent brain, which respectively requires larger concentrations of either calcium or CaM to activate the PM-Ca-ATPase. The observation that the oxidative modification of CaM during normal biological aging results in a reduced calcium sensitivity of the PM-Ca-ATPase, a lower affinity between CaM and the PM-Ca-ATPase, and the reduction in the maximal velocity of the PM-Ca-ATPase is consistent with earlier results that indicate the calcium handling capacity of a range of tissues including brain, heart, and erythrocytes isolated from aged animals declines, resulting in both longer calcium transients and elevated basal levels of intracellular calcium. Thus, the oxidative modification of selected methionines in CaM may explain aspects of the loss of calcium homeostasis associated with the aging process.

We have investigated the ability of methionine sulfoxide reductase (MsrA) to maintain optimal calmodulin (CaM) function through the repair of oxidized methionines, which have been shown to accumulate within CaM in senescent brain [Gao, J., Yin, D. H., Yao, Y., Williams, T. D., and Squier, T. C. (1998) Biochemistry 37, 9536-9548]. Oxidatively modified calmodulin (CaMox) isolated from senescent brain or obtained by in vitro oxidation was incubated with MsrA. This treatment restores the functional ability of CaMox to activate the plasma membrane (PM) Ca-ATPase, confirming that (i) the decreased ability of CaM isolated from senescent animals to activate the PM Ca-ATPase results solely from methionine sulfoxide formation and (ii) MsrA can repair methionine sulfoxides within cytosolic proteins. We have used electrospray ionization mass spectrometry to investigate the extent and rates of methionine sulfoxide repair within CaMox. Upon exhaustive repair by MsrA, there remains a distribution of methionine sulfoxides within functionally reactivated CaMox, which varies from three to eight methionine sulfoxides. The rates of repair of methionine sulfoxides within individual tryptic fragments of CaMox vary by a factor of 2, where methionine sulfoxides located within hydrophobic sequences are repaired in preference to methionines that are more solvent accessible within the native structure. However, no single methionine sulfoxide is completely repaired in all CaM oxiforms. Decreases in the alpha-helical content and a disruption of the tertiary structure of CaM have previously been shown to result from methionine oxidation. Repair of selected methionine sulfoxides in CaMox by MsrA results in a partial refolding of the secondary structure, suggesting that MsrA repairs methionine sulfoxides within unfolded sequences until native-like structure and function are re-attained. The ability of CaMox isolated from senescent brain to fully activate the PM Ca-ATPase following repair by MsrA suggests the specific activity of MsrA is insufficient to maintain CaM function in aging brain. These results are discussed in terms of the possible regulatory role MsrA may play in the modulation of CaM function and calcium homeostasis under conditions of oxidative stress.