Hippocampal long-term potentiation (LTP) induced by theta-burst pairing of Schaffer collateral inputs and postsynaptic firing is associated with localized increases in synaptic strength and dendritic excitability. Using the same protocol, we now demonstrate a decrease in cellular excitability that was blocked by the h-channel blocker ZD7288. This decrease was also induced by postsynaptic theta-burst firing alone, yet it was blocked by NMDA receptor antagonists, postsynaptic Ca2+ chelation, low concentrations of tetrodotoxin, omega-conotoxin MVIIC, calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors and a protein synthesis inhibitor. Increasing network activity with high extracellular K+ caused a similar reduction of cellular excitability and an increase in h-channel HCN1 protein. We propose that backpropagating action potentials open glutamate-bound NMDA receptors, resulting in an increase in I(h) and a decrease in overall excitability. The occurrence of such a reduction in cellular excitability in parallel with synaptic potentiation would be a negative feedback mechanism to normalize neuronal output firing and thus promote network stability.

Asymmetric flow field-flow fractionation (AFFFF) coupled with multiple-angle light scattering (MALS) is a powerful technique showing potential for the analysis of pharmaceutically-relevant virus-like particles (VLPs). A lack of published methods, and concerns that membrane adsorption during sample fractionation may cause sample aggregation, have limited widespread acceptance. Here we report a reliable optimized method for VLP analysis using AFFFF-MALS, and benchmark it against dynamic light scattering (DLS) and transmission electron microscopy (TEM). By comparing chemically identical VLPs having very different quaternary structure, sourced from both bacteria and insect cells, we show that optimized AFFFF analysis does not cause significant aggregation, and that accurate size and distribution information can be obtained for heterogeneous samples in a way not possible with TEM and DLS. Optimized AFFFF thus provides a quantitative way to monitor batch consistency for new vaccine products, and rapidly provides unique information on the whole population of particles within a sample.

Viral self-assembly is of tremendous virological and biomedical importance. Although theoretical and crystallographic considerations suggest that controlled conformational change is a fundamental regulatory mechanism in viral assembly, direct proof that switching alters the thermodynamic attraction of self-assembling components has not been provided. Using the VP1 protein of polyomavirus, we report a new method to quantitatively measure molecular interactions under conditions of rapid protein self-assembly. We show, for the first time, that triggering virus capsid assembly through biologically relevant changes in Ca 2þ concentration, or pH, is associated with a dramatic increase in the strength of protein molecular attraction as quantified by the second virial coefficient (B 22 ). B 22 decreases from 22.3 Â 10 24 mol ml g 22(weak protein -protein attraction) to 22.4 Â 10 23 mol ml g 22 (strong protein attraction) for metastable and Ca 2þ -triggered self-assembling capsomeres, respectively. An assemblydeficient mutant (VP1CD63) is conversely characterized by weak protein -protein repulsion independently of chemical change sufficient to cause VP1 assembly. Concomitant switching of both VP1 assembly and thermodynamic attraction was also achieved by in vitro changes in ammonium sulphate concentration, consistent with protein salting-out behaviour. The methods and findings reported here provide new insight into viral assembly, potentially facilitating the development of new antivirals and vaccines, and will open the way to a more fundamental physico-chemical description of complex protein self-assembly systems.

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Far-infrared (FIR) spectroscopy in the spectral region of 50-450 cm(-1) was used to study a series of protein higher-order structures constructed using β-lactoglobulin and polyomavirus capsid protein VP1. There were marked differences in the spectra for β-lactoglobulin monomer and dimer and between untreated β-lactoglobulin and heat-induced gels formed at neutral pH. Untreated β-lactoglobulin and heat-induced gels formed at acidic pH exhibited little difference in their spectra. Assembly of the quaternary structure of polyomavirus virus-like particles also caused large changes in the FIR spectra. These findings suggest that FIR spectroscopy may prove useful in studying some protein quaternary and higher-order structures. There was evidence of detection of β-lactoglobulin dimerization, intermolecular disulfide bonding in heat-induced neutral gels, and polyomavirus virus-like particle assembly but no evidence that FIR could detect β-lactoglobulin fibrils with their polymeric structure and hydrogen-bonded intermolecular β-pleated sheeting.