Neuronal nicotinic threonine-for-leucine 247 α7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the α7 mutant receptor for 5HT is not modified by the presence of dihydro-β-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates α7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

RNA folding kinetics regulates translation of phage MS2 maturation gene

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem–loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3′ moiety contains the Shine–Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem–loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability

The anomalous temperature dependence of protein folding has received considerable attention. Here we show that the temperature dependence of the folding of protein L becomes extremely simple when the effects of temperature on protein stability are corrected for; the logarithm of the folding rate is a linear function of 1/T on constant stability contours in the temperature–denaturant plane. This convincingly demonstrates that the anomalous temperature dependence of folding derives from the temperature dependence of the interactions that stabilize proteins, rather than from the super Arrhenius temperature dependence predicted for the configurational diffusion constant on a rough energy landscape. However, because of the limited temperature range accessible to experiment, the results do not rule out models with higher order temperature dependences. The significance of the slope of the stability-corrected Arrhenius plots is discussed.

Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding

The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression. Slow promoter binding might be related to TBP’s ability to occlude its DNA binding domain through dimerization. Using a “pull-down” based assay, we find that TBP dimers dissociate slowly (t½ = 6–10 min), and thus present a formidable kinetic barrier to TATA binding. At 10 nM, TBP appears to exist as a mixed population of monomers and dimers. In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers. The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation.

Kinesin’s processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains

Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3–6 s−1 and that detachment from the microtubule occurs at a similar rate (3 s−1). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP⋅Pi state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP⋅Pi state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.

RGS-r, a retinal specific RGS protein, binds an intermediate conformation of transducin and enhances recycling

G proteins regulate intracellular signaling by coupling a cycle of guanine nucleotide binding and hydrolysis to transient changes of cellular functions. The mechanisms that control the recycling of transducin, the “pace-setting” G protein that regulates mammalian phototransduction, are unclear. We show that a novel retinal specific RGS-motif protein specifically binds to an intermediate conformation involved in GTP hydrolysis by transducin and accelerates phosphate release and the recycling of transducin. This specific interaction further rationalizes the kinetics of the phototransduction cascade and provides a general hypothesis to explain the mechanism of interaction of RGS proteins with other G proteins.

Pressure-induced protein-folding/unfolding kinetics

We use an off-lattice minimalist model to describe the effects of pressure in slowing down the folding/unfolding kinetics of proteins when subjected to increasingly larger pressures. The potential energy function used to describe the interactions between beads in the model includes the effects of pressure on the pairwise interaction of hydrophobic groups in water. We show that pressure affects the participation of contacts in the transition state. More significantly, pressure exponentially decreases the chain reconfigurational diffusion coefficient. These results are consistent with experimental results on the kinetics of pressure-denaturation of staphylococcal nuclease.

The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor–epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome–lysosome pathway in these cells.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+ T lymphocytes, i.e., CD4+Ki67+ T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8+Ki67+ T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+ T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+ T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+ T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Zic2 regulates the kinetics of neurulation

Mutation in human ZIC2, a zinc finger protein homologous to Drosophila odd-paired, causes holoprosencephaly (HPE), which is a common, severe malformation of the brain in humans. However, the pathogenesis is largely unknown. Here we show that reduced expression (knockdown) of mouse Zic2 causes neurulation delay, resulting in HPE and spina bifida. Differentiation of the most dorsal neural plate, which gives rise to both roof plate and neural crest cells, also was delayed as indicated by the expression lag of a roof plate marker, Wnt3a. In addition the development of neural crest derivatives such as dorsal root ganglion was impaired. These results suggest that the Zic2 expression level is crucial for the timing of neurulation. Because the Zic2 knockdown mouse is the first mutant with HPE and spina bifida to survive to the perinatal period, the mouse will promote analyses of not only the neurulation but also the pathogenesis of human HPE.

Observation of strange kinetics in protein folding

Highly nonexponential folding kinetics in aqueous solution have been observed during temperature jump-induced refolding of two proteins, yeast phosphoglycerate kinase and a ubiquitin mutant. The observations are most easily interpreted in terms of downhill folding, which posits a heterogeneous ensemble of structures en route to the folded state. The data are also reconciled with exponential kinetics measured under different experimental conditions and with titration experiments indicating cooperative folding.

Magnesium-dependent folding of self-splicing RNA: Exploring the link between cooperativity, thermodynamics, and kinetics

Folding of the Tetrahymena self-splicing RNA into its active conformation involves a set of discrete intermediate states. The Mg2+-dependent equilibrium transition from the intermediates to the native structure is more cooperative than the formation of the intermediates from the unfolded states. We show that the degree of cooperativity is linked to the free energy of each transition and that the rate of the slow transition from the intermediates to the native state decreases exponentially with increasing Mg2+ concentration. Monovalent salts, which stabilize the folded RNA nonspecifically, induce states that fold in less than 30 s after Mg2+ is added to the RNA. A simple model is proposed that predicts the folding kinetics from the Mg2+-dependent change in the relative stabilities of the intermediate and native states.

Stretching single-domain proteins: Phase diagram and kinetics of force-induced unfolding

Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a stepwise “quantized” manner. Unfolding dynamics and forces required to stretch proteins depend sensitively on topology. The unfolding rates increase exponentially with force f till an optimum value, which is determined by the barrier to unfolding when f = 0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single-molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.

Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg2+. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel.