Complex salt bridges in proteins: statistical analysis of structure and function.

We developed an algorithm to analyze the distribution and geometry of simple and complex salt bridges in 94 proteins selected from the Protein Data Bank. In this study, the term "salt bridging" denotes both non-bonded and hydrogen-bonded paired electrostatic interactions between acidic carboxyl groups and basic amino groups in single or adjacent protein chains. We defined complex salt bridges as those joining more than two charged residues, including Asp, Glu, Lys and Arg, and excluding His. The survey related the following special features of complex salt bridges. (1) The abundance of complex salt bridges is high; one-third of all residues participating in salt-bridge formation were part of complex salt bridges. (2) The geometry of the interaction between acidic and basic residues is very similar in simple and complex salt bridges. Adding one residue to a simple interaction represents a minor change in the geometry but provides the molecule with a more complex interaction, a phenomenon that may explain the cooperative effect of salt bridges in proteins. Such moderate changes in salt-bridge networks can be generated stepwise and reversibly without trapping the protein in a local energetic minimum. (3) One important role of complex salt bridges is connecting protein subunits or joining two secondary structures to form quaternary structures, where they can connect as many as five secondary structure units. (4) Arginine serves as a key connector and/or a branching unit because its geometry allows three possible directions of interactions. The information gained from this study of complex salt bridges should enhance the understanding of protein structure.

Continuum solvent model studies of the interactions of an anticonvulsant drug with a lipid bilayer.

Valproic acid (VPA) is a short, branched fatty acid with broad-spectrum anticonvulsant activity. It has been suggested that VPA acts directly on the plasma membrane. We calculated the free energy of interaction of VPA with a model lipid bilayer using simulated annealing and the continuum solvent model. Our calculations indicate that VPA is likely to partition into the bilayer both in its neutral and charged forms, as expected from such an amphipathic molecule. The calculations also show that VPA may migrate (flip-flop) across the membrane; according to our (theoretical) study, the most likely flip-flop path at neutral pH involves protonation of VPA pending its insertion into the lipid bilayer and deprotonation upon departure from the other side of the bilayer. Recently, the flip-flop of long fatty acids across lipid bilayers was studied using fluorescence and NMR spectroscopies. However, the measured value of the flip-flop rate appears to depend on the method used in these studies. Our calculated value of the flip-flop rate constant, 20/s, agrees with some of these studies. The limitations of the model and the implications of the study for VPA and other fatty acids are discussed.

Fusion of LuxA and LuxB and its expression in E. coli, S. cerevisiae and D. melanogaster.

Luciferase from Vibrio harveyi is encoded by two adjacent genes, luxA and luxB. The two genes were fused by replacing a segment extending from near the end of luxA into the N-terminal end of luxB by a synthetic oligonucleotide. The construction removed the TAA stop codon at the end of luxA, the intervening region of 26 base pairs, and the initial methionine of luxB. A Smal site was included at the junction between the two genes and an AatII site was created near the end of luxA without altering its amino acid sequence. In Escherichia coli the fused luxAB gene could be expressed to produce functional luciferase that gave about 20% of the activity in cells without the fusion. An out-of-frame ATG exists close to and preceding the ATG of the luxA gene. This was removed and the entire fused gene bracketed by several restriction enzyme sites. The fused luxAB gene was successfully expressed in Saccharomyces cerevisiae and Drosophila melanogaster by transferring it to appropriate plasmid vectors.