Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers.

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.

G protein activation kinetics and spillover of gamma-aminobutyric acid may account for differences between inhibitory responses in the hippocampus and thalamus.

We have developed a model of gamma-aminobutyric acid (GABA)ergic synaptic transmission mediated by GABAA and GABAB receptors, including cooperativity in the guanine nucleotide binding protein (G protein) cascade mediating the activation of K+ channels by GABAB receptors. If the binding of several G proteins is needed to activate the K+ channels, then only a prolonged activation of GABAB receptors evoked detectable currents. This could occur if strong stimuli evoked release in adjacent terminals and the spillover resulted in prolonged activation of the receptors, leading to inhibitory responses similar to those observed in hippocampal slices. The same model also reproduced thalamic GABAB responses to high-frequency bursts of stimuli. In this case, prolonged activation of the receptors was due to high-frequency release conditions. This model provides insights into the function of GABAB receptors in normal and epileptic discharges.

Negative activation enthalpies in the kinetics of protein folding.

Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.