Kinetic discrimination in T-cell activation.

We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

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.

A combined kinetic and modeling study of the catalytic center subsites of human angiogenin.

Kinetic analysis and molecular modeling have been used to map the ribonucleolytic center of angiogenin (Ang). Pyrimidine nucleotides were found to interact very weakly with Ang, consistent with the inaccessible B1 pyrimidine binding site revealed by x-ray crystallography. Ang also lacks an effective phosphate binding site on the 5' side of B1. Although the B2 site that preferentially binds purines on the 3' side of B1 is also weak, its associated phosphate subsites make substantial contributions: both 3',5'-ADP and 5'-ADP have Ki values 6-fold lower than for 5'-AMP, and adding a 3'-phosphate to the substrate CpA increases Kcat/Km by 9-fold. Thus Ang has a functional P2 site on the 3' side of B2 and a site for a second phosphate on the 5' side of B2. Modeling of an Ang-d(ApTpApA) complex suggested that Arg-5 forms part of the P2 site and that a 2'-phosphate might bind more tightly than a 3'-phosphate. Both predictions were confirmed kinetically. The subsite map obtained by this combined approach indicated that 5'-diphosphoadenosine 2'-phosphate might be a more potent inhibitor than any of the nucleotides tested thus far. Indeed, its Ki value of 150 microM is 50-fold lower than that for the best nucleotide previously reported and 400-fold lower than the Km for the best dinucleotide substrate. This compound may serve as a suitable starting point for the eventual design of tight-binding inhibitors of Ang as antiangiogenic agents for human therapy.

Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique.

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.

Kinetic traps in lysozyme folding.

Folding of lysozyme from hen egg white was investigated by using interrupted refolding experiments. This method makes use of a high energy barrier between the native state and transient folding intermediates, and, in contrast to conventional optical techniques, it enables one to specifically monitor the amount of native molecules during protein folding. The results show that under strongly native conditions lysozyme can refold on parallel pathways. The major part of the lysozyme molecules (86%) refold on a slow kinetic pathway with well-populated partially folded states. Additionally, 14% of the molecules fold faster. The rate constant of formation of native molecules on the fast pathway corresponds well to the rate constant expected for folding to occur by a two-state process without any detectable intermediates. The results suggest that formation of the native state for the major fraction of lysozyme molecules is retarded compared with the direct folding process. Partially structured intermediates that transiently populate seem to be kinetically trapped in a conformation that can only slowly reach the native structure.

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.

Submillisecond folding of monomeric lambda repressor.

The folding kinetics of a truncated form of the N-terminal domain of phage lambda repressor [lambda 6-85] has been investigated by using the technique of dynamic NMR. lambda 6-85 has been shown previously to fold in a purely two-state fashion. This allows the determination of folding and unfolding rates from simulation of the exchange-broadened aromatic resonances of Tyr-22. The folding kinetics were determined over a range of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding rate constants is exponential, suggesting that the rate-determining step is invariant at the urea concentrations studied. The folding and unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400 s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate constant exceeds that of other fast-folding globular proteins by a factor of 14-54. The urea dependence of the folding and unfolding rate constants suggests that the transition state of the rate-determining step is considerably more exposed to solvent than previously studied protein-folding transition states. The surprising rapidity of lambda 6-85 folding and unfolding may be the consequence of its all-helical secondary structure. These kinetic results clearly demonstrate that all of the fundamental events of protein folding can occur on the submillisecond time scale.

Significant discrepancies between van't Hoff and calorimetric enthalpies.

In this paper we show that the usual assumption in studies of the temperature variation of equilibrium constants for equilibria of the form A+B <-->AB that a plot of ln K vs. 1/T (K = equilibrium constant, T = temperature in degrees kelvin) is a straight line with slope equal to -delta HvH/R (delta HvH = van't Hoff or apparent enthalpy, R = gas constant) is not valid in many cases. In all the cases considered here, delta HvH is temperature dependent and is significantly different from the true or calorimetrically measured enthalpy, and the respective values for delta Cp are also significantly different.

NusG is required to overcome a kinetic limitation to Rho function at an intragenic terminator.

Rho-dependent transcription termination at certain terminators in Escherichia coli also depends on the presence of NusG [Sullivan, S. L. & Gottesman, M. E. (1992) Cell 68, 989-994]. We have found that termination at the first intragenic terminator in lacZ (tiZ1) is strongly dependent on NusG when transcription is done in vitro with the concentrations of NTPs found in vivo. With a lower level of NTPs, and consequently a slower rate of RNA-chain growth, Rho causes some termination by itself that is enhanced with NusG. These results suggest that NusG serves to overcome a kinetic limitation of Rho to function at certain terminators. At a second intragenic terminator within the lacZ reading frame (tiZ2) the efficiency of Rho-mediated termination was unaffected by either NusG or by RNA polymerase elongation kinetics. Thus, using purified components and intracellular levels of NTPs, we have confirmed the in vivo finding that certain Rho-dependent terminators also depend on NusG, whereas others do not.

Kinetic proofreading in T-cell receptor signal transduction.

Like other cell-surface receptors with intrinsic or associated protein-tyrosine kinase activity, the T-cell receptor complex undergoes a number of modifications, including tyrosine phosphorylation steps, after ligand binding but before transmitting a signal. The requirement for these modifications introduces a temporal lag between ligand binding and receptor signaling. A model for the T-cell receptor is proposed in which this feature greatly enhances the receptor's ability to discriminate between a foreign antigen and self-antigens with only moderately lower affinity. The proposed scheme is a form of kinetic proofreading, known to be essential for the fidelity of protein and DNA synthesis. A variant of this scheme is also described in which a requirement for formation of large aggregates may lead to a further enhancement of the specificity of T-cell activation. Through these mechanisms, ligands of different affinity potentially may elicit qualitatively different signals.

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.

Electrogenic light reactions in photosystem I: resolution of electron-transfer rates between the iron-sulfur centers.

Flash-induced voltage changes (electrogenic events) in photosystem I particles from spinach, oriented in a phospholipid layer, have been studied at room temperature on a time scale ranging from 1 micros to several seconds. A phospholipid layer containing photosystem I particles was adsorbed to a Teflon film separating two aqueous compartments. Voltage changes were measured across electrodes immersed in the compartments. In the absence of added electron donors and acceptors, a multiphasic voltage increase, associated with charge separation, was followed by a decrease, associated with charge recombination. Several kinetic phases were resolved: a rapid (<1 micros) increase, ascribed to electron transfer from the primary electron donor P700 to the iron-sulfur electron acceptor FB, was followed by a slower, biphasic increase with time constants of 30 and 200 micros. The 30-micros phase is assigned to electron transfer from FB to the iron-sulfur center FA. The voltage decrease had a time constant of 90 ms, ascribed to charge recombination from FA to P700. Upon chemical prereduction of FA and FB the 30- and 200-micros phases disappeared and the decay time constant was accelerated to 330 micros, assigned to charge recombination from the phylloquinone electron acceptor (A1) or the iron-sulfur center FX to P700.

Human autoantibody recognition of DNA.

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.