Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy

High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.     

Effect of pressure on the self-association of melittin

The effect of increased hydrostatic pressure (1 bar to 1.8 kbar) on the self-association of melittin was measured by using the fluorescence anisotropy of its single tryptophan residue. The degree of self-association was found to decrease with increasing pressure. The volume change (delta V) for dissociation is surprisingly large. At low pressures, delta V for dissociation is near -150 mL/mol. The magnitude of the volume change decreased with increasing pressure, possibly as a result of pressure-induced compression of free volume trapped at the subunit interface region of the tetramer. Overall, the pressure-dependent association of melittin is comparable to that expected for hydrophobic interactions and to that found for micelle formation by detergents.     

Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content. The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).     

Histone subunit interactions as investigated by high pressure

High-pressure fluorescence polarization was used to investigate subunit interactions of the histone H2A-H2B dimer and the H3/H4 tetramer isolated from calf thymus (CT) and chicken erythrocyte (CE) chromatin. The proteins were individually labeled with the fluorescent probe 5-(dimethylamino)-naphthalene-1-sulfonate (dansyl or DNS), and the fluorescence polarization was measured as a function of pressure. The long fluorescence lifetime of the probe allows for the observation of global rotations of the protein, the rate of which is dependent upon the aggregation state. From the pressure dependence of the dansyl polarization, the Kd of H2A-H2B dissociation of the CE dimer was found to be approximately 1 X 10(-7) M at 2.0 M NaCl. Lowering the salt concentration to 200 mM slightly stabilized the protein to 6 X 10(-8) M. Our data indicate a small negative volume change for the dissociation of the core particle octamer. The (H3)2(H4)2 tetramer, as was shown in the previous paper (Royer et al., 1989), also formed predominantly dimers of tetramers at higher protein or salt concentrations. In the study presented here, we found the dissociation constant for the H3/H4 octamer to dimer transition to be 1 X 10(-21) M3 (C1/2 = 4 X 10(-8) M) at 2 M NaCl for the CT preparation. Decreasing the salt concentration to 200 mM reduced the stability of the CT H3/H4 octamer to 9 X 10(-21) M3 (C1/2 = 8 X 10(-8) M). The dimer of the CE tetramer also dissociated upon application of pressure in 2 M salt.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

Formation of mixed disulfide adducts at cysteine-281 of the lactose repressor protein affects operator and inducer binding parameters

The lactose repressor protein has been modified with three sulfhydryl-specific reagents which form mixed disulfide adducts. Methyl methanethiosulfonate (MMTS) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) completely reacted with all three cysteine residues, whereas only partial reaction was observed with didansylcystine. Cysteines-107 and -140 reacted stoichiometrically with MMTS and DTNB, while Cys-281 was modified only at higher molar ratios. Didansylcystine reacted primarily with cysteines-107 and -140. Affinity of MMTS-modified repressor for 40 base pair operator DNA was decreased 30-fold compared to unmodified repressor, and this decrease correlated with modification of cysteine-281. DTNB-modified repressor bound operator DNA with a 50-fold weaker affinity than unmodified repressor. Modification of the lac repressor with didanylcystine decreased operator binding only 4-fold, and nonspecific DNA binding increased 3-fold compared to unmodified repressor. No change in the inducer equilibrium binding constant was observed following modification with any of these reagents. In contrast, inducer association and dissociation rate constants were decreased approximately 50-fold for repressor completely modified with MMTS or DTNB, while didansylcystine had minimal effect on inducer binding kinetics. Correlation between modification of Cys-281 and the observed decrease in rate constants indicates that this region of the protein regulates the accessibility of the sugar binding site. The parallel between the increase in the Kd for repressor binding to operator, the altered rate constant for inducer binding, and modification of cysteine-281 suggests that this region of the protein is crucially involved in the function of the repressor protein.     

The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid

The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.     

Radiation abolishes inducer binding to lactose repressor

The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.     

Resolution of the fluorescence decay of the two tryptophan residues of lac repressor using single tryptophan mutants.

We have studied the time-resolved intrinsic tryptophan fluorescence of the lac repressor (a symmetric tetramer containing two tryptophan residues per monomer) and two single-tryptophan mutant repressors obtained by site-directed mutagenesis, lac W201Y and lac W220Y. These mutant repressor proteins have tyrosine substituted for tryptophan at positions 201 and 220, respectively, leaving a single tryptophan residue per monomeric subunit at position 220 for the W201Y mutant and at position 201 in the W220Y mutant. It was found that the two decay rates recovered from the analysis of the wild type data do not correspond to the rates recovered from the analysis of the decays of the mutant proteins. Each of these residues in the mutant repressors displays at least two decay rates. Global analysis of the multiwavelength data from all three proteins, however, yielded results consistent with the fluorescence decay of the wild type lac repressor corresponding simply to the weighted linear combination of the decays from the mutant proteins. The effect of ligation by the antagonistic ligands, inducer and operator DNA, was similar for all three proteins. The binding of the inducer sugar resulted in a quenching of the long-lived species, while binding by the operator decreased the lifetime of the short components. Investigation of the time-resolved anisotropy of the intrinsic tryptophan fluorescence in these three proteins revealed that the depolarization of fluorescence resulted from a fast motion and the global tumbling of the macromolecule. Results from the simultaneous global analysis of the frequency domain data sets from the three proteins revealed anisotropic rotations for the macromolecule, consistent with the known elongated shape of the repressor tetramer. In addition, it appears that the excited-state dipole of tryptophan 220 is alighed with the long axis of the repressor.     

Analysis of trp repressor-operator interaction by filter binding.

A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein.     

Pressure-induced dissociation of solubilized sarcoplasmic reticulum ATPase

The effect of hydrostatic pressure on the self-association of sarcoplasmic reticulum ATPase solubilized by nonionic detergent was studied in the pressure range of 1 atm up to 2 kilobars. Polarization of intrinsic tryptophan fluorescence or of fluorescence of a pyrene probe covalently attached to the ATPase was measured. An increase in hydrostatic pressure promoted dissociation of the protein into monomers. For a midpoint dissociation pressure of 1.3 kilobars, the standard volume change in the dissociation reaction was delta Vop = -167 ml/mol. Full reversibility of the pressure effects was shown to occur, as seen by recovery of polarization. An increase in Ca2+ concentration from 50 microM to 5 mM and of pH from 6.9 to 8.6 were found to increase the midpoint dissociation pressure, indicating that these factors stabilize the dimeric state. The hydrolytic activity of the ATPase was measured under pressure. The activity was inhibited by pressure increase. It was found that an irreversible inactivation of the solubilized enzyme occurred during turnover and that increasing pressure added to this instability. Reversibility of the activity was critically dependent on the presence of 10 mM Ca2+ in the assay medium.     

pH dependence of the dissociation of multimeric hemoglobin probed by high hydrostatic pressure

We investigated the thermodynamic features of the classic alkaline dissociation of multimeric hemoglobin (3.1 MDa) from Glossoscolex paulistus (Annelidea) using high hydrostatic pressure. Light scattering measurements up to microscopic thermodynamic equilibrium indicated a high pH dependency of dissociation and association. Electron microscopy and gel filtration corroborated these findings. The volume change of dissociation decreased in absolute values from -48.0 mL/mol of subunit at pH 6.0 to -19.2 mL/mol at pH 9.0, suggesting a lack of protein interactions under alkaline conditions. Concomitantly, an increase in pH reduced the Gibbs free energy of dissociation from 37.7 to 27.5 kJ/mol of subunit. The stoichiometry of proton release calculated from the pressure-induced dissociation curves was +0.602 mol of H(+)/mol of subunit. These results provide a direct quantification of proton participation in stabilizing the aggregated state of the hemoglobin, and contribute to our understanding of protein-protein interactions and of the surrounding conditions that modulate the process of aggregation.     

Purification and characterization of the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K12

The glpR gene encoding the repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli was cloned downstream from the strong pL promoter of bacteriophage lambda. This allowed overproduction of the repressor upon thermal induction of a cryptic lambda lysogen harboring the cI857 gene. The repressor was purified 40-fold to homogeneity from an induced strain. The purification scheme utilized polyethyleneimine and ammonium sulfate fractionation, followed by phosphocellulose and DEAE-Sephadex chromatography. Purification was monitored by measuring the binding of radiolabeled inducer (sn-glycerol 3-phosphate) to the repressor. The purified repressor migrated as a single band exhibiting a subunit molecular weight of 30,000 assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the repressor under nondenaturing conditions was 100,000-130,000 suggesting the repressor is a tetramer under native conditions. Interaction of the repressor with sn-glycerol 3-phosphate was studied using flow dialysis. Scatchard analysis of the data indicated four binding sites/repressor tetramer and a dissociation constant of 31 microM. Interaction of the repressor with DNA was studied using band-shift electrophoresis. The repressor specifically bound DNA fragments containing the control regions for the glpD, glpK, and glpT-A genes. Binding of DNA by the repressor was diminished in the presence of sn-glycerol 3-phosphate.     

Deletion of lactose repressor carboxyl-terminal domain affects tetramer formation.

The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., and Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Kr?mer, H., and Müller-Hill, B. (1991) New Biol. 3, 57-62). To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed. The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor. The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography. Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins. Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion. Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH. Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer. "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed. The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored. This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure.     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.     

Multiphasic denaturation of the lambda repressor by urea and its implications for the repressor structure.

Urea denaturation of the lambda repressor has been studied by fluorescence and circular dichroic spectroscopies. Three phases of denaturation could be detected which we have assigned to part of the C-terminal domain, N-terminal domain and subunit dissociation coupled with further denaturation of the rest of the C-terminal domain at increasing urea concentrations. Acrylamide quenching suggests that at least one of the three tryptophan residues of the lambda repressor is in a different environment and its emission maximum is considerably blue-shifted. The transition in low urea concentration (midpoint approximately 2 M) affects the environment of this tryptophan residue, which is located in the C-terminal domain. Removal of the hinge and the N-terminal domain shifts this transition towards even lower urea concentrations, indicating the presence of interaction between hinge on N-terminal and C-terminal domains in the intact repressor.     

Energy transfer in lactose repressor protein modified with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate

Energy transfer between the two tryptophan residues in the lactose repressor protein and the fluorescent moiety of the cysteine-specific reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (1,5-IAEDANS) has been examined. Modification of repressor with this compound did not affect operator or inducer binding. 1,5-IAEDANS reacted primarily with Cys140 in wild-type repressor [Schneider et al. (1984) Biochemistry 23, 2221]; in the presence of inducer, modification at Cys107 increased, while reaction at Cys140 remained unchanged. Energy transfer between tryptophans and the AEDANS moiety(ies) in wild-type lac repressor occurred with an efficiency of 6.7 +/- 1.9% in the absence and 7.8 +/- 1.6% in the presence of inducer. The distance between the Trp donor(s) and the acceptor in wild-type repressor was calculated to be in the range approximately 35 A under both conditions. The similarity in efficiency despite large differences in the amount of acceptor attached to Cys107 when inducer is bound indicates that the AEDANS group at position 107 does not participate significantly in energy transfer and that the label at position 140 acts as the primary acceptor group. The similarity of energy-transfer efficiency (7.1 +/- 3.8%) observed for 1,5-IAEDANS-modified monomeric mutant repressor (Y282D) indicates that the transfer is primarily intrasubunit in the native tetramer. Measurements using two mutant repressors (each with a single tryptophan and modified with 1,5-IAEDANS) demonstrated that both tryptophans can serve as donor in the energy-transfer process. The W201Y repressor (containing Trp220) exhibited a transfer efficiency lower than wild type (5.6 +/- 2.4%), corresponding to a slightly larger distance between the donor-acceptor pair in this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and modification of a monomeric mutant of the lactose repressor protein

A monomeric mutant lactose repressor protein (T-41), containing serine at position 282 in place of tyrosine [Schmitz, A., Schmeissner, U., Miller, J. H., & Lu, P. (1976) J. Biol. Chem. 251, 3359-3366], has been purified by a series of chromatographic and precipitation methods. The molecular weight of the mutant as determined by gel filtration was approximately 40,000. The inducer equilibrium binding constant for the mutant was comparable to that of the tetrameric wild-type repressor at pH 7.5, whereas operator DNA binding was not detectable. In contrast to wild-type repressor, equilibrium and kinetic rate constants for inducer binding to the monomer were largely independent of pH; thus, the quaternary structure of the wild-type repressor is required for the pH-associated effects on inducer binding. Although ultraviolet absorbance difference spectra indicated that inducer binding to T-41 protein elicited different changes in the environment of aromatic residues from those generated in wild-type repressor, the shift in the fluorescence emission maximum in response to inducer binding was similar for T-41 and wild-type repressors. Similarity in 1-anilinonaphthalene-8-sulfonic acid binding to monomer and tetramer suggests that this fluorophore does not bind at subunit interfaces. Modification of Cys-281 with methyl methanethiosulfonate was observed at low molar ratios of reagent per T-41 monomer (4-fold). This result is in contrast to data observed for tetrameric wild-type repressor which requires high molar ratios for this cysteine to react. We conclude that Cys-281, adjacent to the site of the T-41 mutation, is located on the surface of the monomer in this region crucial for subunit interaction.