Re-evaluation of the role of thiol groups in rabbit muscle aldolase A.

Exposed thiol groups of rabbit muscle aldolase A were modified by 5,5'-dithiobis(2-nitrobenzoic) acid with concomittant loss of enzyme activity. When 5-thio-2-nitrobenzoate residues bound to enzyme SH groups were replaced by small and uncharged cyanide residues the enzyme activity was restored by more than 50%. The removal of a bulky C-terminal tyrosine residue from the active site of aldolase A resulted in enzyme which was inhibited by 5,5'-dithiobis(2-nitrobenzoic) acid only by 50% and its activity was nearly unchanged after modification of its thiol groups with cyanide. The results obtained show directly that rabbit muscle aldolase A does not possess functional cysteine residues and that the inactivation of the enzyme caused by sulfhydryl group modification reported previously can be attributed most likely to steric hindrance of a catalytic site by modifying agents.

The reactivity and function of cysteine residues in rabbit liver aldolase B.

Rabbit liver aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) contains 8 SH groups/subunit and no disulfide bonds. In the native enzyme 3 SH groups/subunit are titrable with 5,5'-dithiobis(2-nitrobenzoic) acid (Nbs2), 2,2'-dithiodipyridine and N-ethylmaleimide, whereas p-mercuribenzoate is able to react with 4 thiol groups per subunit. Among the three thiol groups titrable with Nbs2, two react 'fast' with simple second-order kinetics, one reacts 'slow' and for this thiol group saturation kinetics is observed, suggesting a reversible binding of Nbs2 to the enzyme prior to covalent modification. It is shown that this binding most likely occurs via ionic interactions in the region close to the active site. The kinetic differentiation between the two 'fast' reacting groups is possible by kinetic analysis of the release of Nbs residues from the modified enzyme. Modification of all exposed SH groups of aldolase B results in 14-32% loss of enzymatic activity. The complete inactivation of liver aldolase by 1 mM p-mercuribenzoate reported previously (Waud, J.M., Feldman, E. and Schray, K.J. (1981) Arch. Biochem. Biophys. 206, 292-295) is shown to be caused by a nonspecific reaction of this reagent used in large excess. It is concluded that this isoenzyme differs from muscle aldolase in the reactivity of exposed SH groups, the mechanisms of the interaction with modifying agents and also in the effect of SH group modification on the enzymatic activity.

HMG1 proteins from evolutionary distant organisms distort B-DNA conformation in similar way.

The abundant high-mobility group proteins 1/2 (HMG1/2) represent a group of potent architectural elements of chromatin. They are able to induce strong bends and untwist DNA. Here, we compared the abilities of diverse HMG1 proteins to distort the B-DNA conformation of 30-base pair DNA fragment. The DNA bending was measured in solution by monitoring fluorescence resonance energy transfer between fluorescence probes attached to opposite ends of the DNA fragment. Various insect and plant proteins which differ in size, in composition of their HMG1-box domains (HMG1-BD), and in composition of the N- and the C-terminally flanking regions were analyzed in these experiments. Despite these structural differences the extent of the induced changes in DNA conformation upon binding to various proteins was similar, as the estimated bend angle was 150+/-20 degrees for all the tested proteins. Our results suggest that a set of highly conserved residues stabilizing the tertiary structure of the HMG1-BD mainly determines the extent of DNA bending in the complex. Even extended positively charged regions flanking the HMG1-BD are apparently not able to influence this conformational distortion of DNA.

DNA-induced conformational changes in cyclic AMP receptor protein: detection and mapping by a protein footprinting technique using multiple chemical proteases.

Cyclic AMP receptor protein (CRP) is a regulator of transcription in Escherichia coli which mediates its activity by binding specific DNA sequences in a cyclic AMP-dependent manner. The interaction of CRP with specific DNA was probed by a protein footprinting technique using chemical proteases of different charge, size, and hydrophobicity. The experimental data were compared with known crystal structures of cAMP-CRP and cAMP-CRP-DNA complexes to determine a correlation between the structure of the complexes, the nature of the chemical protease and protein cleavage patterns. In addition, such comparison allowed us to determine if DNA binding in solution induced conformational changes in the protein not apparent in the crystal structure. In the cAMP-CRP-DNA complex, both the protections and the enhancements of proteolytic cleavage were observed outside of the known CRP-DNA interface, suggesting that CRP undergoes a conformational change upon binding DNA. Among the observed changes, the most interesting were those around the B alpha-helix and beta-strand 8, since this region overlaps with the activation region 2 which CRP uses for protein-protein interactions with RNA polymerase. DNA-induced changes were observed also in the region involved in CRP-CytR interaction and in CRP intersubunit contact regions. These data suggest that binding of DNA in solution induces conformational changes in CRP which can be transmitted via intersubunit contacts to regions of the protein involved in interactions with other members of transcriptional machinery.

Determinants for Escherichia coli RNA polymerase assembly within the beta subunit.

We used binding assays and other approaches to identify fragments of the Escherichia coli RNAP beta subunit involved in the obligatory interaction with the alpha subunit to form the stable assembly intermediate alpha2beta as well as in the interaction to recruit the beta' subunit into the alpha2beta sub-assembly. We show that two regions of evolutionarily conserved sequence near the C terminus of beta (conserved regions H and I) are central to the assembly of RNAP and likely make subunit-subunit contacts with both alpha and beta'.

Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.

CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830-10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA]2-), iron complexed to ethylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine mesylate ([Fe-HDFO]+), and copper complexed to o-phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA] and [Fe-DTPA]2- of CRP-cAMP detected hypersensitivities in the DNA-binding F alpha-helix, the interdomain hinge, and the ends of the C alpha-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA]2- also detected reductions in cleavage in the D and E alpha-helices, which are involved in DNA recognition. Cleavage by [Fe-HDFO]+ of CRP-cAMP detected hypersensitivities in beta-strand 8, the B alpha-helix, as well as in parts of the F and C alpha-helices. [Fe-HDFO]+ also detected protections from cleavage in beta-strands 4 to 5 and their intervening loop, beta-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E alpha-helices. Cleavage by [(OP)2Cu]+ of CRP-cAMP detected hypersensitivities in beta-strands 9 and 11 as well as in the D and E alpha-helices. [(OP)2Cu]+ also detected protections in the C alpha-helix , the interdomain hinge, and beta-strands 2-7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)2Cu]+, which detected minor protections in beta-strands 3-7, the interdomain hinge, and the C alpha-helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction. Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone.

Luminescence resonance energy transfer analysis of RNA polymerase complexes.

Resonance energy transfer allows measurement of atomic-scale distances under a variety of solution conditions. Luminescence resonance energy transfer (LRET) is a variant of energy transfer measurement in which lanthanide chelates are used as the probes. The unusual properties of lanthanide emission, in particular their long microsecond-scale lifetimes, offer several advantages for energy transfer measurements with biological samples. One of the unique features of LRET is the ability to measure energy transfer under conditions where severe heterogeneity of labeled macromolecules exists. This feature of LRET is the special emphasis of this article. We describe here LRET methodology with a particular attention to using sensitized acceptor emission to determine efficiency of energy transfer. Although we employed this technique in the characterization of Escherichia coli RNA polymerase complexes it is readily compatible with the study of essentially any protein.

Luminescence energy transfer with lanthanide chelates: interpretation of sensitized acceptor decay amplitudes.

Lanthanide chelates used as donors offer several advantages over classical fluorescence probes in resonance energy transfer distance measurements. One of these advantages is that energy transfer can be conveniently measured using sensitized acceptor decay measurements. In these measurements a long microsecond lifetime of the lanthanide donor and a short nanosecond lifetime of the acceptor allow elimination of a signal from the unquenched donor. Therefore, the decay of sensitized acceptor emission reflects decay properties of the donor engaged in energy transfer. The purpose of this work is to point out the importance of the fact that the amplitude of the sensitized acceptor signal is dependent on the resonance energy transfer rate constant. Thus, in the case where there are two or more populations of donors with different energy transfer rate constants, the relative amplitudes of corresponding decay components observed in sensitized acceptor emission do not represent the relative populations of the donors. We use simulations to show that these effects can be very significant. A minor population of donors with a high rate of energy transfer can produce sensitized acceptor decay which is dominated by a decay component corresponding to this minor donor population. Using a simple experimental system of rapid diffusion limit energy transfer between a europium chelate and Cy5 acceptor we show that the predicted dependency of sensitized acceptor decay amplitude on the energy transfer rate is indeed observed. We suggest that the relative importance of decay components observed in sensitized acceptor emission should be evaluated after an appropriate correction of their values such that they properly reflect possible different populations of donors. We describe a method to perform such correction.

Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach.

A novel direct approach, analogous to DNA footprinting, for mapping protein domains involved in macromolecular interactions is presented in this paper and applied to cAMP receptor protein (CRP) interactions with the allosteric ligand (cAMP) and DNA. In this approach, a protein-macromolecule complex is subjected to a nonspecific cleavage by Fe-EDTA. The cleavage products are resolved by SDS-PAGE and transferred to a PVDF membrane. Transferred polypeptides are visualized by immunostaining with antibodies specific to the N-terminal peptide of the protein. The mobility of the bands visualized in such a way is directly proportional to the distance of the cleavage sites from the N-terminus, and thus the positions of the sites protected from cleavage by a bound macromolecule can be determined. Thus, protein domains involved in macromolecular interactions can be mapped. In the case of CRP, the cleavage conditions were established which resulted in, on the average, less than one cleavage event/protein molecule and which preserved satisfactory levels of protein and DNA activity. When applied to CRP-DNA interactions, the protein footprinting approach correctly identified domains of CRP that were known to be involved in the recognition of DNA. The obtained results showed also that the binding of CRP to the DNA binding site perturbed the region of CRP involved in intersubunit interactions. An allosteric ligand (cAMP) appeared to perturb the same region of CRP. This stresses out the importance of intersubunit interactions in cAMP modulation of protein DNA binding affinity. The protein footprinting methodology presented in this paper should be broadly generalizable to any protein-macromolecule system.

Physical studies on interaction of transcription activator and RNA-polymerase: fluorescent derivatives of CRP and RNA polymerase.

Protein-protein interactions between cAMP receptor protein (CRP) and RNA polymerase (RNAP) have been proposed to be essential in RNAP activation by CRP in type I promoters. These two proteins were shown to interact in solution in the absence of promoter DNA (Heyduk et al., 1993). In this report we describe the preparation of fluorescent derivatives of CRP (fluorescent probes at position 13 and 85); and of the alpha-subunit of RNAP (at position 321). The specific incorporation of fluorescence probes was achieved by expressing protein in a bacteria strain, auxotrophic for tryptophan, in media containing 5-hydroxytryptophan (5-OH-Trp). The absorbance spectrum of a protein containing 5-OH-Trp is shifted towards longer wavelengths as compared to the native protein. This allows selective monitoring of the fluorescence signal of 5-OH-Trp derivative of a protein even in the presence of high concentration of tryptophan containing protein(s). The CRP derivative is shown to retain 100% of the native protein cAMP binding and specific DNA binding activity. Using a fluorescence polarization assay, it is also shown that 5-OH-Trp derivative of CRP interacts with RNAP as well as the native protein. The RNAP reconstituted with 5-OH-Trp derivative of the alpha-subunit retained the enzymatic activity. Fluorescence quenching studies show that Trp 321 of alpha-subunit is located in the region of the protein which is exposed to a solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiol-reactive, luminescent Europium chelates: luminescence probes for resonance energy transfer distance measurements in biomolecules.

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using luminescence resonance energy transfer measurements [P. R. Selvin, T. M. Rana, and J. E. Hearst (1994) J. Am. Chem. Soc. 116, 6029-6030; P. R. Selvin, and J. E. Hearst (1994) Proc. Natl. Acad. Sci. USA 91, 10024-10028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds contain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. These reagents can be used to label proteins at Cys residues or synthetic oligonucleotides which contain thiol groups. Modification can be reversed easily by treatment with a reducing agent (dithiothreitol). Luminescence energy transfer between these new chelates and CY5 fluorochrome attached to the opposite ends of 15-bp double-stranded DNA was measured to test their usefulness for distance measurements in macromolecules. The distance measured between the chelate (donor) and CY5 (acceptor) was in the range expected for the length of 15-bp DNA. The stability of europium chelates and their conjugates with a protein, the precision of distance measurements using these chelates, possible errors due to intramolecular energy transfer, and the modulation of the R0 value with deuterium oxide were tested. The results obtained fully confirmed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence resonance energy transfer.

Mapping conformational changes in a protein: application of a protein footprinting technique to cAMP-induced conformational changes in cAMP receptor protein.

We have used protein footprinting [Heyduk, E., & Heyduk, T. (1994) Biochemistry 33, 9643] to detect and map ligand-induced conformational changes in cAMP receptor protein (CRP). The binding of cAMP to CRP dramatically increases the specific DNA binding activity of the protein and, as has been previously shown, induces conformational changes in the protein. Protein footprinting experiments with the free CRP, the CRP-cAMP complex, and the CRP-cGMP complex were analyzed quantitatively. Binding of cAMP produced measurable differences in the susceptibility of CRP to the cleavage by Fe-EDTA. Almost all of these changes occurred in the C-terminal domain (DNA binding domain) of the protein. Additional changes were observed at the ends of the C alpha-helix, which is involved in intersubunit contacts in the CRP dimer, and in the hinge peptide, connecting N-terminal and C-terminal domains of the protein. The boundaries of the regions in the C-terminal domain, which exhibited changes in susceptibility to Fe-EDTA cleavage, almost exactly corresponded to D, E, and F alpha-helices which are involved directly in the recognition of DNA. The F alpha-helix, which provides all base-specific contacts in the CRP-DNA complex, became hypersensitive to Fe-EDTA-mediated cleavage, whereas the solvent exposure of D and E alpha-helices was decreased upon binding of cAMP. These results suggest that a significant part of cAMP-induced conformational change in CRP involves a movement of secondary structure elements in the C-terminal domain of the protein so that the recognition F alpha-helix becomes exposed to the solvent. In contrast to cAMP, binding of cGMP produced insignificant changes in susceptibility to Fe-EDTA-mediated cleavage. This is consistent with the inability of cGMP to induce functional conformational changes in CRP. The protein footprinting technique appears to be sufficiently sensitive for detection and mapping of ligand-induced conformational changes in proteins.

Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study.

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme. Five single reactive cysteine mutants of sigma70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled sigma70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma70 is located in an alpha-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.

Conformation and DNA binding properties of a single-stranded DNA binding region of sigma 70 subunit from Escherichia coli RNA polymerase are modulated by an interaction with the core enzyme.

A derivative of the sigma 70 subunit from Escherichia coli RNA polymerase with specific fluorescence probes in conserved region 2.3 (DNA "melting motif") was prepared by replacing tryptophan residues at positions 314 and 326 of the wild-type sigma 70 with alanine. The remaining two tryptophan residues (Trp 433 and 434) of [Ala 314, 326]sigma 70 were biosynthetically replaced with 5-hydroxy-tryptophan (5OHTrp), a fluorescent tryptophan analogue with unique emission that can be selectively observed both in free 5OHTrp[Ala 314, 326]sigma 70 as well as in 5OHTrp[Ala 314, 326]sigma 70 bound to the core RNA polymerase. Fluorescence quenching experiments revealed that positions 433 and 434 were solvent exposed in free 5OHTrp[Ala314, 326]sigma 70. The binding of sigma 70 to core polymerase reduced the solvent exposure of these residues. In the presence of single-stranded oligonucleotides, fluorescence of 5OHTrp at position 433 and 434 was quenched approximately 65% and these residues became inaccessible to the solvent. Using fluorescence of 5OHTrp at positions 433 and 434 as a specific signal of DNA binding, we show that free sigma 70 bound single-stranded DNA weakly and did not discriminate between nontemplate and template strand of promoter DNA. Binding of sigma 70 to the core increased the affinity for binding nontemplate DNA, whereas the affinity to template or "nonspecific" DNA was reduced, resulting in a holoenzyme which could bind nontemplate strand approximately 200-fold better then the template strand. We concluded that Trp 433 and 434 of sigma 70 are located within a single-stranded DNA binding region of sigma 70 and that binding of sigma 70 to the core enzyme induced conformational changes in a single-stranded DNA binding region of the protein. As a consequence of these conformational changes, sigma 70 subunit gains the specificity for the nontemplate strand of the melted region in the "open" complex.

Temperature-induced conformational transition in rabbit muscle aldolase studied by temperature dependence of sulfhydryl reactivity.

A temperature-induced non-denaturing conformational transition in rabbit muscle aldolase has been as subject of discussion and controversy for some period of time. In this study the temperature dependence of the reactivity of aldolase SH groups is investigated in order to detect subtle changes in the enzyme conformation. For model thiol-containing systems such as cysteine, glutathione and bovine serum albumin, linear Arrhenius plots have been obtained for the reaction with 5,5'-dithiobis(2-nitrobenzoic acid). On the other hand, for rabbit muscle pyruvate kinase, a protein which undergoes temperature-induced conformational transition, the plot obtained is nonlinear with a break at the temperature (18 degrees C) close to that reported earlier. In the case of aldolase the Arrhenius plots for three slowly reacting SH groups (Cys-72, 289, 338) and a fast reacting group (Cys-239) are nonlinear with a break at about 26-27 degrees C. The fluorescence measurements show that a plot of the fluorescence intensity of tryptophan residues versus temperature exhibits a break at the same temperature. It is shown that the observed conformational change is fully reversible. In the presence of the competitive inhibitor hexitol 1,6-bisphosphate, which is known to protect Cys-72 and Cys-338 from chemical modification, the Arrhenius plot exhibits a break for the fast reacting Cys-239 residue and is linear for the slowly reacting Cys-289. It is found that 0.6 M urea increases the transition temperature for all exposed SH groups of aldolase. The above results show that at several points in the aldolase molecule, including the active-site region, an abrupt change of microenvironments takes place with temperature. The competitive inhibitor protects a portion of aldolase molecule against the thermal transition.

Long-range effects and conformational flexibility of aldolase.

The conformational flexibility and long-range interactions in rabbit muscle aldolase induced by active-site ligand binding, cross-linking of the enzyme between Cys72 and Cys338, and removal of the C-terminal tyrosine residue were studied by following the changes in the microenvironments of Cys239 and Cys289 located outside the active site. It was found that substrates induced a conformational change in aldolase, which propagates from the active site to Cys239, which is located close to intersubunit contacts. The response of the enzyme is differential. Ligands having both C-1 and C-6 phosphates or C-1 phosphate only induce the enhancement of Cys239 reactivity, whereas those with C-6 phosphates only decrease Cys239 reactivity. This correlates well with a dramatic difference in kinetic parameters for a cleavage of fructose-1,6-P2 and fructose-1-P. Therefore, these changes can be interpreted as syncatalytic. Cross-linking of the aldolase subunit by an -S-S-bridge between Cys72 and Cys338 inactivates the enzyme, abolishes binding of active-site ligands, and induces a conformational change in the enzyme that can be detected far away (at Cys239 and Cys289) from the site of perturbation. Cys72 and Cys338 are not in the active site. This shows that the region of the active site and the environment of Cys72 and Cys338 are tightly coupled and that residues far away from the active site, through such coupling, can possess properties of active-site residues. Similar, although less dramatic changes are observed upon removal of the C-terminal tyrosine residue. In view of the results obtained in this paper, aldolase seems to be quite a flexible molecule, whose conformation is sensitive to the nature of a substrate bound to the enzyme and is able to transmit the information about a local perturbation over long distances within a molecule.

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities.

Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.

Solution studies on the structure of bent DNA in the cAMP receptor protein-lac DNA complex.

Cyclic AMP receptor protein is involved in the regulation of more than 20 genes. A step in the mechanism of activation of transcription is to induce a significant bending of the DNA upon complex formation between specific DNA and the protein. The induced DNA bending and a structure of the protein-DNA complex were studied by fluorescence energy transfer in 50 mM Tris, 1 mM EDTA, and 50 mM KCl at pH 7.8 and 20 degrees C. The symmetry of the DNA bend was estimated by measuring the efficiency of transfer between the protein and a label on either the upstream or the downstream end of a lac DNA fragment. The results show that the bend, despite the asymmetry in the DNA sequence, is symmetrical, for the fragments which length ranges from 26 to 40 bp. Using fluorescence energy transfer, the extent of DNA bending was estimated by measuring the end-to-end distance of the DNA fragment which was labeled with a donor-acceptor pair on two opposite ends. Both steady-state and time-resolved measurements showed that in a 26 bp lac DNA fragment complexed with cyclic AMP receptor protein, the end-to-end distance is about 77 A which corresponds to a bending angle of 80 degrees or 100 degrees, depending on the actual contour length between the fluorophores in the free DNA fragment. The results using longer DNA fragments show no measurable amount of energy transfer; thus, it is very unlikely that the DNA completely wraps around the CRP molecule.(ABSTRACT TRUNCATED AT 250 WORDS)