Universality classes in folding times of proteins

Molecular dynamics simulations in simplified models allow one to study the scaling properties of folding times for many proteins together under a controlled setting. We consider three variants of the Go models with different contact potentials and demonstrate scaling described by power laws and no correlation with the relative contact order parameter. We demonstrate existence of at least three kinetic universality classes that are correlated with the types of structure: the alpha-, alpha-beta-, and beta- proteins have the scaling exponents of approximately 1.7, 2.5, and 3.2, respectively. The three classes merge into one when the contact range is truncated at a reasonable value. We elucidate the role of the potential associated with the chirality of a protein.     

Kinetic nonoptimality and vibrational stability of proteins

Scaling of folding times in Go models of proteins and of decoy structures with the Lennard-Jones potentials in the native contacts reveal power law trends when studied under optimal folding conditions. The power law exponent depends on the type of native geometry. Its value indicates lack of kinetic optimality in the model proteins. In proteins, mechanical and thermodynamic stabilities are correlated.     

Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization,degradation, and mRNA destabilization

Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.     

Temporal genetic variation in Aedes aegypti populations in Ho Chi Minh City (Vietnam)

Aedes aegypti, the main vector of dengue viruses in Asia, displays variation in population density over time. The larval habitats of this species being unevenly distributed and transient (depending on cycles of drought and flood), the forces generating temporal variation in gene frequencies in populations are studied. We sampled seven mosquito populations from Ho Chi Minh City (Vietnam) and its suburbs on five occasions between April 1999 and August 2000. We investigated genetic variation by studying isoenzyme and microsatellite polymorphism and susceptibility to a dengue 2 virus strain. Ae. aegypti populations collected during the dry season (January-April) showed genetic differentiation (F(ST) = 0.016, P < 10(-6) for isoenzymes) and showed more differentiated infection rates of the dengue 2 virus. The genetic structure of the population is less marked during the rainy season (F(ST) = 0.081, P < 10(-6)). Thus, environmental factors, such as rainfall and factors related to human activity, such as breeding site density and insecticide treatment, control the genetic structure of Ae. aegypti populations in the short term. The implications of studies of this kind for the design of future control programmes are discussed.     

Recombinant equine cytochrome c in Escherichia coli: high-level expression,characterization, and folding and assembly mutants

To promote studies of cytochrome c (Cyt c) ranging from apoptosis to protein folding, a system for facile mutagenesis and high-level expression is desirable. This work used a generally applicable strategy for improving yields of heterologously expressed protein in Escherichia coli. Starting with the yeast Cyt c plus heme lyase construct of Pollock et al. [Pollock, W. B., Rosell, F. I., Twitchett, M. B., Dumont, M. E., and Mauk, A. G. (1998) Biochemistry 37, 6124-6131], an E. coli-based system was designed that consistently produces high yields of recombinant eucaryotic (equine) Cyt c. Systematic changes to the ribosome binding site, plasmid sequence, E. coli strain, growth temperature, and growth duration increased yields from 2 to 3 mg/L to as much as 105 mg/L. Issues related to purification, fidelity of heme insertion, equilibrium stability, and introduction and analysis of mutant forms are described. As an example, variants tailored for folding studies are discussed. These remove known pH-dependent kinetic folding barriers (His26 and His33 and N-terminus), reveal an additional kinetic trap at higher pH due to some undetermined residue(s), and show how a new barrier can be placed at different points in the folding pathway in order to trap and characterize different folding intermediates. In addition, destabilizing glycine mutants in the N-terminal helix are shown to affect the fractional yield of a heme inverted Cyt c isoform.     

Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.     

Activation of Metabotropic Glutamate Receptors Inhibits Synapsin I Phosphorylation in Visceral Sensory Neurons

Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca⁺⁺ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mm KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-α-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia. Received 28 June 2000/Revised: 11 September 2000     

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.