Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

The kinetics of photo-induced electrontransfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferarfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of 4.8 x 10(7) M(-1) x s(-1). The limiting value of kobs at high HiPIP concentration is 95 s(-1). The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is approximately 3 x 10(5) s(-1). This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 microM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 10(6) M(-1) x s(-1) and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferar fermentans HiPIP is a very effective electron donor to the photosynthetic RC.

Nonuniformity of the constitutive law parameters for shear rupture and quasistatic nucleation to dynamic rupture: a physical model of earthquake generation processes.

Based on the recent high-resolution laboratory experiments on propagating shear rupture, the constitutive law that governs shear rupture processes is discussed in view of the physical principles and constraints, and a specific constitutive law is proposed for shear rupture. It is demonstrated that nonuniform distributions of the constitutive law parameters on the fault are necessary for creating the nucleation process, which consists of two phases: (i) a stable, quasistatic phase, and (ii) the subsequent accelerating phase. Physical models of the breakdown zone and the nucleation zone are presented for shear rupture in the brittle regime. The constitutive law for shear rupture explicitly includes a scaling parameter Dc that enables one to give a common interpretation to both small scale rupture in the laboratory and large scale rupture as earthquake source in the Earth. Both the breakdown zone size Xc and the nucleation zone size L are prescribed and scaled by Dc, which in turn is prescribed by a characteristic length lambda c representing geometrical irregularities of the fault. The models presented here make it possible to understand the earthquake generation process from nucleation to unstable, dynamic rupture propagation in terms of physics. Since the nucleation process itself is an immediate earthquake precursor, deep understanding of the nucleation process in terms of physics is crucial for the short-term (or immediate) earthquake prediction.

Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism.

We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

Kinetics of cytokine expression during primary human immunodeficiency virus type 1 infection.

In the present study, we have determined the kinetics of constitutive expression of a panel of cytokines [interleukin (IL) 2, IL-4, IL-6, IL-10, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha)] in sequential peripheral blood mononuclear cell samples from nine individuals with primary human immunodeficiency virus infection. Expression of IL-2 and IL-4 was barely detected in peripheral blood mononuclear cells. However, substantial levels of IL-2 expression were found in mononuclear cells isolated from lymph node. Expression of IL-6 was detected in only three of nine patients, and IL-6 expression was observed when transition from the acute to the chronic phase had already occurred. Expression of IL-10 and TNF-alpha was consistently observed in all patients tested, and levels of both cytokines were either stable or progressively increased over time. Similar to IL-10 and TNF-alpha, IFN-gamma expression was detected in all patients; however, in five of nine patients, IFN-gamma expression peaked very early during primary infection. The early peak in IFN-gamma expression coincided with oligoclonal expansions of CD8+ T cells in five of six patients, and CD8+ T cells mostly accounted for the expression of this cytokine. These results indicate that high levels of expression of proinflammatory cytokines are associated with primary infection and that the cytokine response during this phase of infection is strongly influenced by oligoclonal expansions of CD8+ T cells.

The nucleation of receptor-mediated endocytosis.

A theory of the mechanical origins of receptor-mediated endocytosis shows that a spontaneous membrane complex formation can provide the stimulus for a local membrane motion toward the cytosol. This motion is identified with a nucleation stage of receptor-mediated endocytosis. When membrane complexes cluster, membrane deformation is predicted to be most rapid. The rate of growth of membrane depressions depends upon the relative rates of approach of aqueous cytosolic and extracellular fluids toward the cell membrane. With cytosolic and extracellular media characterized by apparent viscosities, the rate of growth of membrane depressions is predicted to increase as the extracellular viscosity nears the apparent viscosity of the cytosol and then to decrease when the extracellular viscosity exceeds that of the cytosol. To determine whether these trends would be apparent in the overall endocytosis rate constant, an experimental study of transferrin-mediated endocytosis in two different cell lines was conducted. The experimental results reveal the same dependence of internalization rate on extracellular viscosity as predicted by the theory. These and other comparisons with experimental data suggest that the nucleation stage of receptor-mediated endocytosis is important in the overall endocytosis process.

Correlation between kinetics and RNA splicing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in neocortical neurons.

In the cortex fast excitatory synaptic currents onto excitatory pyramidal neurons and inhibitory nonpyramidal neurons are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors exhibiting cell-type-specific differences in their kinetic properties. AMPA receptors consist of four subunits (GluR1-4), each existing as two splice variants, flip and flop, which critically affect the desensitization properties of receptors expressed in heterologous systems. Using single cell reverse transcription PCR to analyze the mRNA of AMPA receptor subunits expressed in layers I-III neocortical neurons, we find that 90% of the GluR1-4 in nonpyramidal neurons are flop variants, whereas 92% of the GluR1-4 in pyramidal neurons are flip variants. We also find that nonpyramidal neurons predominantly express GluR1 mRNA (GluR1/GluR1-4 = 59%), whereas pyramidal neurons contain mainly GluR2 mRNA (GluR2/GluR1-4 = 59%). However, the neuron-type-specific splicing is exhibited by all four AMPA receptor subunits. We suggest that the predominance of the flop variants contributes to the faster and more extensive desensitization in nonpyramidal neurons, compared to pyramidal cells where flip variants are dominant. Alternative splicing of AMPA receptors may play an important role in regulating synaptic function in a cell-type-specific manner, without changing permeation properties.

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.

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Gain and kinetics of activation in the G-protein cascade of phototransduction.

The guanine nucleotide binding protein (G protein) cascade underlying phototransduction is one of the best understood of all signaling pathways. The diffusional interactions of the proteins underlying the cascade have been analyzed, both at a macroscopic level and also in terms of the stochastic nature of the molecular contacts. In response to a single activated rhodopsin (R*) formed as a result of a single photon hit, it can be shown that molecules of the G-protein transducin will be activated approximately linearly with time. This, in turn, will cause the number of activated molecules of the effector protein (the phosphodiesterase) also to increase linearly with time. These kinetics of protein activation provide an accurate description of the time course of the rising phase of the photoreceptor's electrical response over a wide range of flash intensities. Recent estimates indicate that at room temperature each R* triggers activation of the phosphodiesterase at a rate of 1000-2000 subunits.s-1. Now that a quantitative description of the activation steps in transduction has been obtained, perhaps the greatest challenge for the future is to provide a comprehensive description of the shutoff reactions, so that a complete account of the photoreceptor's response to light can be achieved.

Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.

Optimization of rates of protein folding: the nucleation-condensation mechanism and its implications.

Small, single-module proteins that fold in a single cooperative step may be paradigms for understanding early events in protein-folding pathways generally. Recent experimental studies of the 64-residue chymotrypsin inhibitor 2 (CI2) support a nucleation mechanism for folding, as do some computer stimulations. CI2 has a nucleation site that develops only in the transition state for folding. The nucleus is composed of a set of adjacent residues (an alpha-helix), stabilized by long-range interactions that are formed as the rest of the protein collapses around it. A simple analysis of the optimization of the rate of protein folding predicts that rates are highest when the denatured state has little residual structure under physiological conditions and no intermediates accumulate. This implies that any potential nucleation site that is composed mainly of adjacent residues should be just weakly populated in the denatured state and become structured only in a high-energy intermediate or transition state when it is stabilized by interactions elsewhere in the protein. Hierarchical mechanisms of folding in which stable elements of structure accrete are unfavorable. The nucleation-condensation mechanism of CI2 fulfills the criteria for fast folding. On the other hand, stable intermediates do form in the folding of more complex proteins, and this may be an unavoidable consequence of increasing size and nucleation at more than one site.

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.

Kinetics of spindle pole body separation in budding yeast.

In the budding yeast Saccharomyces cerevisiae, the spindle pole body (SPB) serves as the microtubule-organizing center and is the functional analog of the centrosome of higher organisms. By expressing a fusion of a yeast SPB-associated protein to the Aequorea victoria green fluorescent protein, the movement of the SPBs in living yeast cells undergoing mitosis was observed by fluorescence microscopy. The ability to visualize SPBs in vivo has revealed previously unidentified mitotic events. During anaphase, the mitotic spindle has four sequential activities: alignment at the mother-daughter junction, fast elongation, translocation into the bud, and slow elongation. These results indicate that distinct forces act upon the spindle at different times during anaphase.

Simple model of protein folding kinetics.

A simple model of the kinetics of protein folding is presented. The reaction coordinate is the "correctness" of a configuration compared with the native state. The model has a gap in the energy spectrum, a large configurational entropy, a free energy barrier between folded and partially folded states, and a good thermodynamic folding transition. Folding kinetics is described by a master equation. The folding time is estimated by means of a local thermodynamic equilibrium assumption and then is calculated both numerically and analytically by solving the master equation. The folding time has a maximum near the folding transition temperature and can have a minimum at a lower temperature.

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.