Effects of CMAP and electrostatic cutoffs on the dynamics of an integral membrane protein: the phospholamban study.

In our effort to understand the microscopic structure and dynamics of phospholamban (PLB), a small integral membrane protein, we have performed a series of 5-20 ns molecular dynamics simulations to explore the influence of environment (solution and lipid bi-layer) and force field (CMAP correction and Ewald summation) on the protein behavior. Under all simulation conditions, we have observed the same major features: existence of two well-defined helical domains at the N- and C-termini, and large-amplitude rigid-body motions of these domains. The average inter-helix angle of PLB was sensitive to the environment. In the methanol and water solution trajectories, the two helical domains tended to adopt a closed orientation, with the inter-helix angle below 90 degrees, while in the lipid bi-layer the domains tend to be in an open conformation, with the inter-helix angle above 90 degrees. Within each studied environment, simulations employing different force field models provided qualitatively similar description of PLB structure and dynamics. The only significant discrepancy was the presence of pi-helical hydrogen bonds in trajectories generated with the standard CHARMM22 force field. Simulations with the CMAP correction, with both cutoff and Ewald electrostatics, exhibited predominantly alpha-helical and some 3(10)-helical hydrogen bonding interactions, and no pi-helical hydrogen bonding, in accord with NMR data. Thus, our results indicate that models including CMAP, with both cutoff and Ewald electrostatics, provide the most realistic description of PLB structure and dynamics. Results obtained from these simulations are in a good agreement with the experimental observables. These include helical secondary structure of PLB, the range explored by the inter-helix angle in methanol, as well as the inter-helix distance and C-terminal helix orientation in the DPPC bi-layer. The observed effect of opening up of the PLB inter-helix angle in the lipid environment relative to solution is also qualitatively reproduced in the simulations, as is the more rigid and compact structure of the C-terminal domain in the membrane relative to solution. The populations of conformations with relatively open inter-domain angles, as well as large fluctuations of this coordinate in DPPC bi-layers allow the N-terminal helix to come into contact with the PLB binding site on the calcium ATPase. Additionally, the presence of a twisting motion around the helical axis enables the helix to orient the correct face to the binding site. Another interesting observation is that the phosphorylation sites Ser(16) and Thr(17) are essentially always accessible to solvent, and presumably also to phosphorylation.

Structure and dynamics of calcium-activated calmodulin in solution.

Two 4-ns molecular dynamics simulations of calcium loaded calmodulin in solution have been performed, using both standard nonbonded cutoffs and Ewald summation to treat electrostatic interactions. Our simulation results are generally consistent with solution experimental studies of calmodulin structure and dynamics, including NMR, cross-linking, fluorescence and x-ray scattering. The most interesting result of the molecular dynamics simulations is the detection of large-scale structural fluctuations of calmodulin in solution. The globular N- and C-terminal domains tend to move approximately like rigid bodies, with fluctuations of interdomain distances within a 7 A range and of interdomain angles by up to 60 deg. Essential dynamics analysis indicates that the three dominant types of motion involve bending of the central helix in two perpendicular planes and a twist in which the domains rotate in opposite directions around the central helix. In the more realistic Ewald trajectory the protein backbone remains mostly within a 2-3 A root-mean-square distance from the crystal structure, the secondary structure within the domains is conserved and middle part of the central helix becomes disordered. The central helix itself exhibits limited fluctuations, with its bend angle exploring the 0-50 degrees range and the end-to-end distance falling in 39-43 A. The results of the two simulations were similar in many respects. However, the cutoff trajectory exhibited a larger deviation from the crystal, loss of several helical hydrogen bonds in the N-terminal domain and lack of structural disorder in the central helix.

Fast kinetics and mechanisms in protein folding.

This review describes how kinetic experiments using techniques with dramatically improved time resolution have contributed to understanding mechanisms in protein folding. Optical triggering with nanosecond laser pulses has made it possible to study the fastest-folding proteins as well as fundamental processes in folding for the first time. These include formation of alpha-helices, beta-sheets, and contacts between residues distant in sequence, as well as overall collapse of the polypeptide chain. Improvements in the time resolution of mixing experiments and the use of dynamic nuclear magnetic resonance methods have also allowed kinetic studies of proteins that fold too fast (greater than approximately 10(3) s-1) to be observed by conventional methods. Simple statistical mechanical models have been extremely useful in interpreting the experimental results. One of the surprises is that models originally developed for explaining the fast kinetics of secondary structure formation in isolated peptides are also successful in calculating folding rates of single domain proteins from their native three-dimensional structure.

A convenient synthesis of 5beta-cholestan-26-oic and 5beta-cholestan-26,27-dioic acids.

A new method for the preparation of 5beta-cholestan-26-oic acids 7 and their analogs is described. The key steps in the synthesis are: iodination of bis- and tris-formyloxy-5beta-cholan-24-ols 3; nucleophilic substitution of iodides 4 with diethyl sodiomalonate; complete alkaline hydrolysis of esters 5; and subsequent decarboxylation of geminal diacids 6 in DMSO.

Aphidicolin glycinate inhibits human neuroblastoma cell growth in vivo.

Aphidicolin is a fungal derived tetracyclic diterpene antibiotic. It is selectively toxic for neuroblastoma (NB) cells in vitro but has no significant effects on the viability of normal human cells and a variety of other tumor entities. We evaluated the antitumoral effects of the water soluble ester aphidicolin glycinate (AphiG) on established human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. Furthermore, we explored the efficacy of direct intraneoplastic and systemic delivery of AphiG. Systemic administration of AphiG (60 mg/kg intraperitoneally, twice per day on 10 consecutive days) significantly suppressed tumor growth but was not able to induce any cures. In contrast, intratumoral AphiG injections (60 or 40 mg/kg/twice a day for 4 days) induced complete tumor regression. Two weeks after the end of treatment no tumor cells were microscopically detectable. Animals were free of tumor for more than 90 days. Histologic examination of inner organs and bone marrow did not reveal any apparent toxic effects of AphiG. These data strongly indicate that AphiG deserves further evaluation as a specific treatment for neuroblastoma.

Oxidative modification of a carboxyl-terminal vicinal methionine in calmodulin by hydrogen peroxide inhibits calmodulin-dependent activation of the plasma membrane Ca-ATPase.

In order to investigate the possibility that calmodulin (CaM) may be a principal target of reactive oxygen species (ROS) produced under conditions of oxidative stress, we have examined wheat germ CaM for the presence of highly reactive sites that correlate with the loss of function. Using reversed-phase HPLC and FAB mass spectrometry after proteolytic digestion, we have identified the sites of modification by hydrogen peroxide. We find that one of the vicinal methionines (i.e., Met146 or Met147) near the C-terminus of CaM is selectively oxidized. The ability of CaM to bind and to activate the plasma membrane (PM)-Ca-ATPase from erythrocytes was measured. There is a 30-fold decrease in the calcium affinity of oxidatively modified CaM. While there is a little change in the binding constant between the carboxyl-terminal domain of calcium-saturated CaM and a peptide homologous to the autoinhibitory sequence of the PM-Ca-ATPase, we find that there is a 9-fold reduction in the affinity of the amino-terminal domain of CaM with respect to the ability to bind target peptides. The extent of oxidative modification to one of the vicinal methionines near the carboxyl-terminal domain correlates with the loss of CaM-dependent activation of the PM-Ca-ATPase. The presence of oxidatively modified CaM prevents native CaM from activating the PM-Ca-ATPase, indicating that the oxidatively modified CaM binds to the autoinhibitory sequence on the Ca-ATPase in an altered nonproductive conformation. We suggest that the functional sensitivity of CaM to the oxidation of one of the C-terminal vicinal methionines permits CAM to serve a regulatory role in modulating cellular metabolism under conditions of oxidative stress. The predominant oxidation of a methionine near the carboxyl terminal of CaM is rationalized in terms of the enhanced solvent accessibility of these vicinal methionines.