Optimum bacteriocin production by Lactobacillus plantarum 17.2b requires absence of NaCl and apparently follows a mixed metabolite kinetics

Bacteriocins from lactic acid bacteria are ribosomally synthesized anti-microbial compounds that may find applications from food preservation to healthcare. Food preservation, using in situ bacteriocin production is the most obvious and simple. Frequently, the best conditions for bacteriocin production are those prevailing during food fermentation but a better understanding of the relationship between growth and bacteriocin production is required. In this work, we evaluate the effects of some environmental factors on bacteriocin production by Lactobacillus plantarum 17.2b. A first screening design showed that NaCl, temperature, pH and the type and concentration of carbon and nitrogen sources were most influents. A moderate stimulatory effect of ethanol and oleuropein was also registered. Two consecutive central composite designs were used to examine the effect of the selected variables and to compute its optimum. The evolution of changes produced by the alterations in environmental factors was further examined trough perturbation plots. Bacteriocin production by L. plantarum 17.2b was very sensitive to environmental conditions and uncoupled from growth. Maximum production required suboptimal growth temperatures, pH values above growth's optimum and no NaCl. A preliminary approach to kinetics showed that bacteriocin production by this strain apparently follows mixed metabolite kinetics.     

Kinetics of pH-dependent interactions between PD-1 and PD-L1 immune checkpoint proteins from molecular dynamics

Immune checkpoint blockade of signaling pathways such as PD-1/PD-L1 has recently opened up a new avenue for highly efficient immunotherapeutic strategies to treat cancer. Since tumor microenvironments are characterized by lower pH (5.5-7.0), pH-dependent protein-ligand interactions can be exploited as efficient means to regulate drug affinity and specificity for a variety of malignancies. In this article, we investigate the mechanism and kinetics of pH-dependent binding and unbinding processes for the PD-1/PD-L1 checkpoint pair employing classical molecular dynamics simulations. Two representative pH levels corresponding to circumneutral physiological conditions of blood (pH 7.4) and acidic tumor microenvironment (pH 5.5) are considered. Our calculations demonstrate that pH plays a key role in protein-ligand interactions with small pH changes leading to several orders of magnitude increase in binding affinity. By identifying the binding pocket in the PD-1/PD-L1 complex, we show a pivotal role of the His68 protonation state of PD-1in the complex stabilization at low pH. The results on the reaction rate constants are in qualitative agreement with available experimental data. The obtained molecular details are important for further engineering of binding/unbinding kinetics to formulate more efficient immune checkpoint blockade strategies.     

Kinetics of the initial steps of rabbit psoas myofibrillar ATPases studied by tryptophan and pyrene fluorescence stopped-flow and rapid flow-quench. Evidence that cross-bridge detachment is slower than ATP binding

The kinetics of the tryptophan fluorescence enhancement that occurs when myofibrils (rabbit psoas) are mixed with Mg-ATP were studied by stopped-flow in different solvents (water, 40% ethylene glycol, 20% methanol) at 4 degrees C. Under relaxing conditions (low Ca(2+)) in water (mu = 0.16 M, pH 7.4) and at high ATP concentrations, the transient was biphasic, giving a k(fast)(max) of 230 s(-)(1) and a k(slow)(max) of 15 s(-)(1). The kinetics of the two phases were compared with those obtained by chemical sampling using [gamma-(32)P]ATP and quenching in acid (P(i) burst experiments: these give unambiguously the ATP cleavage kinetics), or cold Mg-ATP (cold ATP chase: ATP binding kinetics). k(slow) is due to ATP cleavage, as with S1. Interestingly, k(fast) is slower than the ATP binding kinetics. Instead, this constant appears to report ATP-induced cross-bridge detachment from actin because (1) it was identical to the fluorescence transient obtained on addition of ATP to pyrene-labeled myofibrils; (2) when the initial filament overlap in the myofibrils was decreased, the amplitude of the fast phase decreased; (3) there was no fluorescent enhancement upon the addition of ADP to myofibrils. This is different from the situation with S1 or actoS1 where there was also a fast fluorescent ATP-induced transient but whose kinetics were identical to those of the tight ATP binding. To increase the time resolution and to confirm our results, we also carried out transient kinetics in ethylene glycol and methanol. We interpret our results by a scheme in which a rapid equilibrium between attached (AM.ATP) and detached (M.ATP) states is modulated by the fraction of myosin heads in rigor (AM) during the time of experiment.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.     

Iron-histidine stretching vibration in the deoxy state of insect hemoglobins with different O2 affinities and Bohr effects

Resonance Raman spectroscopy has been employed to detect the iron-proximal histidine stretching mode in deoxyhemoglobins from insect larvae of Chironomus thummi thummi (CTT). With the excitation of 413.1 nm, we observe a sharp and intense line in the 220-224 cm-1 region. The assignment of this line to the Fe-N epsilon (His) stretching mode was made on the basis of a 3-cm-1 shift upon 57Fe/54Fe isotope substitution. The Fe-N epsilon (His) vibration is used to monitor the possible changes in the Fe-N epsilon (His) bond strength (hence bone length) in the deoxy state of the monomeric (CTT I, III, and IV) and dimeric (CTT II) insect hemoglobins. As these hemoglobins differ in O2 affinity, off-rate and on-rate constants, and in the Bohr effect, they are excellent model systems for investigating the mechanism of protein control of the heme reactivity. Some of these hemoglobins (CTT III, IV, and II) are allosteric, exhibiting two interconvertible conformational states with high and low O2 affinity at high and low pH, respectively. The Fe-N epsilon (His) stretching frequency does not correlate with the O2 affinity, the on-rate and the off-rate constants for different hemoglobins, for different conformational states, and for modified hemoglobins with different heme peripheral groups. This vibrational mode is insensitive to deuteration of the heme vinyl groups. It is important to note that the Fe-N epsilon (His) bonds in the high pH (high-affinity) and the low pH (low-affinity) states are identical. This implies that the O2 molecule, prior to binding, "sees" identical binding sites. Thus, the difference in free energy changes upon O2 binding is manifested only in the oxy form.     

Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60–91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91–230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).     

The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.     

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.     

Fragment length influences affinity for Cu2+ and Ni2+ binding to His96 or His111 of the prion protein and spectroscopic evidence for a multiple histidine binding only at low pH

The prion protein (PrP) is a Cu2+-binding cell-surface glycoprotein. Using various PrP fragments and spectroscopic techniques, we show that two Cu2+ ions bind to a region between residues 90 and 126. This region incorporates the neurotoxic portion of PrP, vital for prion propagation in transmissible spongiform encephalopathies. Pentapeptides PrP-(92-96) and PrP-(107-111) represent the minimum motif for Cu2+ binding to the PrP-(90-126) fragment. Consequently, we were surprised that the appearance of the visible CD spectra for two fragments of PrP, residues 90-126 and 91-115, are very different. We have shown that these differences do not arise from a change in the co-ordination geometry within the two fragments; rather, there is a change in the relative preference for the two binding sites centred at His111 and His96. These preferences are metal-, pH- and chain-length dependent. CD indicates that Cu2+ initially fills the site at His111 within the PrP-(90-126) fragment. The pH-dependence of the Cu2+ co-ordination is studied using EPR, visible CD and absorption spectroscopy. We present evidence that, at low pH (5.5) and sub-stoichiometric amounts of Cu2+, a multiple histidine complex forms, but, at neutral pH, Cu2+ binds to individual histidine residues. We have shown that changes in pH and levels of extracellular Cu2+ will affect the co-ordination mode, which has implications for the affinity, folding and redox properties of Cu-PrP.     

Simulation of electrostatic and hydrodynamic properties of Serratia endonuclease

We analyze the electrostatic and hydrodynamic properties of a nuclease from the pathogenic gram-negative bacterium Serratia marcescens using finite-difference Poisson-Boltzmann methods for electrostatic calculations and a bead-model approach for diffusion coefficient calculations. Electrostatic properties are analyzed for the enzyme in monomeric and dimeric forms and also in the context of DNA binding by the nuclease. Our preliminary results show that binding of a double-stranded DNA dodecamer by nuclease causes an overall shift in the charge of the protein by approximately three units of elementary charge per monomer, resulting in a positively charged protein at physiologic pH. In these calculations, the free enzyme was found to have a negative (−1 e) charge per monomer at pH 7. The most dramatic shift in pK_a involves His 89 whose pK_a increases by three pH units upon DNA binding. This shift leads to a protonated residue at pH 7, in contrast to the unprotonated form in the free enzyme. DNA binding also leads to a decrease in the energetic distances between the most stable protonation states of the enzyme. Dimerization has no significant effect on the electrostatic properties of each of the monomers for both free enzyme and that bound to DNA. Results of hydrodynamic calculations are consistent with the dimeric form of the enzyme in solution. The computed translational diffusion coefficient for the dimer model of the enzyme is in very good agreement with measurements from light scattering experiments. Preliminary electrooptical calculations indicate that the dimer should possess a large dipole moment (approximately 600 Debye units) as well as substantial optical anisotropy (limiting reduced linear electric dichroism of about 0.3). Therefore, this system may serve as a good model for investigation of electric and hydrodynamic properties by relaxation electrooptical experiments. © 1997 John Wiley & Sons, Inc.     

Determination of solution conformations of PrP106-126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics.

Experimental two-dimensional 1H NMR data have been obtained for PrP106-128 under the following solvent conditions: deionized water/2, 2,2-trifluoroethanol 50 : 50 (v/v) and dimethylsulfoxide. These data were analyzed by restrained molecular mechanics calculations to determine how changes in solvation affect the conformation of the peptide. In deionized water at pH 3.5, the peptide adopted a helical conformation in the hydrophobic region spanning residues Met112-Leu125, with the most populated helical region corresponding to the Ala115-Ala119 segment ( approximately 10%). In trifluoroethanol/H2O, the alpha-helix increased in population especially in the Gly119-Val122 tract ( approximately 25%). The conformation of this region was found to be remarkably sensitive to pH, as the Ala120-Gly124 tract shifted to an extended conformation at pH 7. In dimethylsulfoxide, the hydrophobic cluster adopted a prevalently extended conformation. For all tested solvents the region spanning residues Asn108-Met112 was present in a 'turn-like' conformation and included His111, situated just before the starting point of the alpha-helix. Rather than by conformational changes, the effect of His111 is exerted by changes in its hydrophobicity, triggering aggregation. The amphiphilic properties and the pH-dependent ionizable side-chain of His111 may thus be important for the modulation of the conformational mobility and heterogeneity of PrP106-126.     

The pKa value of His 57-Asp 102 couple in the active site of bovine pancreatic beta-trypsin: a molecular orbital study

The charge relay hypothesis generated a large number of theoretical and experimental studies that tested the ideas involved. Opinion based upon theoretical and experimental studies is divided on the prediction, although there are many experimental data which do not support the hypothesis. The essential feature is the proton transfer from the histidine imidazole to the aspartate. Thus, we have performed the detailed calculations of the proton transfer from His 57 to Asp 102 including the environment of the couple in protonated bovine pancreatic β-trypsin. The charge state of the His 57-Asp 102 couple is greatly influenced by the environment of the enzyme around it. In this paper, it is shown that the proton between His 57 and Asp 102 is covalently bonded to the His 57 imidazole in the protonated β-trypsin. Our MO calculations, which support the neutral-pK-histidine theory as the results, do not support the charge relay mechanism.     

Structural Basis of pH Dependence of Neoculin,a Sweet Taste-Modifying Protein

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.     

Kinetics of folding and unfolding of goat alpha-lactalbumin

The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters.     

The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent pH-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of human cathepsin B by its propeptide is defined by slow binding kinetics with a Ki of 3.7 nM and at pH 4.0 by classical kinetics with a Ki of 82 nM. This pH dependency is essentially eliminated either by the removal of a portion of the enzyme's occluding loop through deletion mutagenesis or by the mutation of either residue Asp22 or His110 to alanine; e.g., the mutant enzyme His110Ala is inhibited by its propeptide with Ki's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His110Ala mutant the inhibition also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzyme, and it has been shown that this bridge stabilizes the occluding loop in its closed position [N?gler, D. K., et al. (1997) Biochemistry 36, 12608-12615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propeptide. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing the occluding loop in its closed conformation, the loop more effectively competes with the propeptide than at higher pH where deprotonation of His110 and the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thus suggesting a possible influence of occluding loop stability on the rate of processing.     

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.     

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.     

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.     

Correlation between binding rate constants and individual information of E. coli Fis binding sites

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.