DNase I footprint analysis of protein-DNA binding.

Deoxyribonuclease I (DNase I) protection mapping, or footprinting, is a valuable technique for locating the specific binding sites of proteins on DNA. The basis of this assay is that bound protein protects the phosphodiester backbone of DNA from DNase I-catalyzed hydrolysis. Binding sites are visualized by autoradiography of the DNA fragments that result from hydrolysis, following separation by electrophoresis on denaturing DNA sequencing gels. Footprinting has been developed further as a quantitative technique to determine separate binding curves for each individual protein-binding site on the DNA. For each binding site, the total energy of binding is determined directly from that site's binding curve. For sites that interact cooperatively, simultaneous numerical analysis of all the binding curves can be used to resolve both the intrinsic binding and cooperative components of these energies.DNase I footprint titration is described in this unit and involves (1) preparation of a singly end-labeled DNA restriction fragment, (2) equilibration of the protein with DNA, (3) exposure of the equilibrium mixture to DNase I, and (4) electrophoretic separation on gels of the denatured hydrolysis products, followed by autoradiography. A describes (1) densitometric analysis of the autoradiograms to obtain binding data and (2) numerical analysis of the binding data to yield binding curves and equilibrium constants for the interactions at each of the separate sites. An describes the qualitative use of footprinting to identify DNA-binding proteins in crude extracts.

Role of multiple CytR binding sites on cooperativity, competition, and induction at the Escherichia coli udp promoter.

The CytR repressor fulfills dual roles as both a repressor of transcription from promoters of the Escherichia coli CytR regulon and a co-activator in some circumstances. Transcription is repressed by a three-protein complex (cAMP receptor protein (CRP)-CytR-CRP) that is stabilized by cooperative interactions between CRP and CytR. However, cooperativity also means that CytR can recruit CRP and, by doing so, can act as a co-activator. The central role of cooperativity in regulation is highlighted by the fact that binding of the inducer, cytidine, to CytR is coupled to CytR-CRP cooperativity; this underlies the mechanism for induction. Similar interactions at the different promoters of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism but also provide differential expression of these genes. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. Recently, we showed that CytR binds specifically to multiple sites in the E. coli deoP promoter, thereby providing competition for CRP binding to CRP operator site 1 (CRP1) and CRP2 as well as cooperativity. The effect of the competition at this promoter is to negate the role of CytR in recruiting CRP. Here, we have used quantitative footprint and mobility shift analysis to investigate CRP and CytR binding to the E. coli udp promoter. Here too, we find that CytR both cooperates and competes for CRP binding. However, consistent with both the distribution of CytR recognition motifs in the sequence of the promoter and the regulation of the promoter, the competition is limited to CRP2. When cytidine binds to CytR, the effect on cooperativity is very different at the udp promoter than at the deoP2 promoter. Cooperativity with CRP at CRP1 is nearly eliminated, but the effect on CytR-CRP2 cooperativity is negligible. These results are discussed in relation to the current structural model of CytR in which the core, inducer-binding domain is tethered to the helix-turn-helix, DNA-binding domain via flexible peptide linkers.

Analysis of protein and DNA-mediated contributions to cooperative assembly of protein-DNA complexes.

The cooperative assembly of protein-DNA complexes is a widespread phenomenon that is of particular significance to transcriptional regulation. Assembly of these complexes is controlled by the chemistry of the macromolecular interactions. In this sense, transcriptional regulation is a chemical issue. The purpose of this review is to present an analytical approach designed to understand this regulation from a chemical perspective. By investigating the solution interactions between all combinations of molecules, protein-protein, protein-ligand, and protein-DNA, and the interplay between them, it is possible to determine the relative free energies of the different configurations of the regulatory complex. This governs their distribution and thereby controls the biological activity. To illustrate the approach, we will address the molecular basis for cooperativity in the bacteriophage lambda, lysogenic-lytic switch mechanism, a system that has long served as a paradigm for gene regulation. The driving force for cooperativity in the assembly of gene regulatory complexes is generally thought to be provided by direct protein-protein interactions. However, other interactions mediated by both proteins and DNA are also involved and may be critical to the regulatory mechanism. We will review advances over the past several years in the application of biophysical chemical methods to investigate protein-protein and protein-DNA interactions. Many of these applications were first employed for the lambda system. In addition to describing the physical basis for the methods, we will focus on the unique information that can be gained and how to combine the information obtained from several techniques to develop a comprehensive view of the critical regulatory interactions.

Activation of gene expression by a ligand-induced conformational change of a protein-DNA complex.

IlvY protein binds cooperatively to tandem operator sites in the divergent, overlapping, promoter-regulatory region of the ilvYC operon of Escherichia coli. IlvY positively regulates the expression of the ilvC gene in an inducer-dependent manner and negatively regulates the transcription of its own divergently transcribed structural gene in an inducer-independent manner. Although binding of IlvY protein to the tandem operators is sufficient to repress ilvY promoter-specific transcription, it is not sufficient to activate transcription from the ilvC promoter. Activation of ilvC promoter-specific transcription requires the additional binding of a small molecule inducer to the IlvY protein-DNA complex. The binding of inducer to IlvY protein does not affect the affinity of IlvY protein for the tandem operator sites. It does, however, cause a conformational change of the IlvY protein-DNA complex, which is correlated with the partial relief of an IlvY protein-induced bend of the DNA helix in the ilvC promoter region. This structural change in the IlvY protein-DNA complex results in a 100-fold increase in the affinity of RNA polymerase binding at the ilvC promoter site. The ability of a protein to regulate gene expression by ligand-responsive modulation of a protein-DNA structure is an emerging theme in gene regulation.

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.

Allosteric mechanism of induction of CytR-regulated gene expression. Cytr repressor-cytidine interaction.

Transcription from cistrons of the Escherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member. A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA. Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified. Each has a substitution for Asp281 or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.

An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor.

Sequence specific DNA binding by regulatory proteins provides the basis for regulation of initiation of transcription. A great deal of progress has been made toward understanding sequence specific recognition by individual protein subunits. An additional level of control that needs to be understood is that due to coupling between the subunits of oligomeric regulatory proteins. An example is the bacteriophage lambda cI repressor, a dimeric protein that regulates the lysogenic to lytic genetic switch of the phage. Two levels of specificity are critical to this regulation. First, like all transcriptional regulators, dimers distinguish operator from nonspecific DNA. Direct readout of the DNA sequence by the recognition helix is considered the well understood mechanism for this. However, differential affinity for O(R)1, O(R)2 and O(R)3 is equally critical to the switch because it mediates opposing regulation of divergent promoters. Site specificity at this second level is less well understood. Conformational adaptation by both the repressor and the different operators appears to be important. To evaluate how subunit-subunit interactions are involved in this process, we investigated the effects on both dimer stability and operator binding of amino acid substitutions at the contacts between the symmetrically related helices-5 in the dimer interface. Substitutions for Tyr88 alter dimer stability and greatly perturb differential operator affinity, but generally do not affect operator versus non-operator specificity. The pattern of these effects suggests that the geometry of the face-to-face aromatic stacking interaction between symmetrically related Tyr88 in each subunit, a group in the dimer interface but far removed from the DNA binding interface, plays a critical role in operator discrimination. Conformational changes in the tertiary structure of the subunits appears to be involved. By contrast, the significant effect of I84S substitution is to greatly decrease affinity for all three operators. Presumably, the altered packing of the dimer interface causes a quarternary structural change that moves the two helix-turn-helix motifs out of register with successive DNA major grooves.

Multiple specific CytR binding sites at the Escherichia coli deoP2 promoter mediate both cooperative and competitive interactions between CytR and cAMP receptor protein.

Binding of cAMP receptor protein (CRP) and CytR mediates both positive and negative control of transcription from Escherichia coli deoP2. Transcription is activated by CRP and repressed by a multi-protein CRP.CytR.CRP complex. The latter is stabilized by cooperative interactions between CRP and CytR. Similar interactions at the other transcriptional units of the CytR regulon coordinate expression of the transport proteins and enzymes required for nucleoside catabolism. A fundamental question in both prokaryotic and eukaryotic gene regulation is how combinatorial mechanisms of this sort regulate differential expression. To understand the combinatorial control mechanism at deoP2, we have used quantitative footprint and gel shift analysis of CRP and CytR binding to evaluate the distribution of ligation states. By comparison to distributions for other CytR-regulated promoters, we hope to understand the roles of individual states in differential gene expression. The results indicate that CytR binds specifically to multiple sites at deoP2, including both the well recognized CytR site flanked by CRP1 and CRP2 and also sites coincident with CRP1 and CRP2. Binding to these multiple sites yields both cooperative and competitive interactions between CytR and CRP. Based on these findings we propose that CytR functions as a differential modulator of CRP1 versus CRP2-mediated activation. Additional high affinity specific sites are located at deoP1 and near the middle of the 600-base pair sequence separating P1 and P2. Evaluation of the DNA sequence requirement for specific CytR binding suggests that a limited array of contiguous and overlapping CytR sites exists at deoP2. Similar extended arrays, but with different arrangements of overlapping CytR and CRP sites, are found at the other CytR-regulated promoters. We propose that competition and cooperativity in CytR and CRP binding are important to differential regulation of these promoters.

Steady-state and time-resolved phosphorescence of wild-type and modified bacteriophage λcI repressors.

We have measured the steady-state phosphorescence and decay times of wild-type λcI repressor and compared it with that of a modified λcI repressor in which > 95% of the tryptophans were replaced with 5-hydroxy-L-tryptophan (5-OHTrp). The wild-type and 5-OHTrp-λcI repressors are spectroscopically distinct such that we can selectively excite the 5-OHTrp-λcI even in the presence of a 15-fold molar excess ofN-acetyltryptophanamide (NATrpA). The phosphorescence band of wild-type λcI is red-shifted by 3 nm relative to NATrpA, characteristic of buried tryptophan. Similarly, the phosphorescence of 5-OHTrp-λcI repressor is red-shifted relative to the model, 5-OHTrp, showing that according to the phosphorescence, the modified repressor is structurally indistinguishable from the native repressor. While the phosphorescence decay of both NATrpA and 5-OHTrp are single exponentials, the decay of both wild-type and 5-OHTrp-λcI repressors is complex, requiring three decay components whose fractional contributions to the phosphorescence are the same for both repressors. Because the 5-OHTrp phosphorescence can be excited at wavelengths outside the absorbance range of tryptophan and DNA, a protein spectrally enhanced with this emitter will aid the investigations of protein-protein or protein-DNA interactions.

Effects of anomalous migration and DNA to protein ratios on resolution of equilibrium constants from gel mobility-shift assays.

Numerical resolution of binding constants from data generated by the gel mobility-shift assay is dependent on the experimental resolution of the different ligation states of the DNA. Previously we showed that the populations of the intermediate ligation states at partial saturation with the protein ligand are extremely sensitive to cooperativity (Senear, D. F. and Brenowitz, M. J. Biol. Chem. 1991, 266, 13661-13671). This makes accurate gel mobility-shift data extremely useful to the demonstration of cooperativity. However, the accuracy with which the intermediate ligation state populations are resolved has been questioned. Thus, two additional and related questions are now considered. First, what information is available if the intermediate ligation state populations are not used in the analysis of binding constants. Second, is accurate information obtained from those states under conditions of high DNA concentration. These questions are addressed by using the interactions of the lambda cI repressor protein with the three site operator, OR, and the interaction of the E. coli GalR protein with the single site operator, OE. Both simulated and experimental data are analyzed. The results point to two conclusions. First, precise resolution of all macroscopic constants for binding of proteins to DNA is critically dependent on the intermediate ligation state populations; resolution is limited to at most two DNA sites if these states are not used in the analysis. Second, when the DNA and protein concentrations used in the titrations are comparable, the resolution of binding constants is extremely sensitive to experimental uncertainty in the macromolecule concentrations.

The primary self-assembly reaction of bacteriophage lambda cI repressor dimers is to octamer.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

5-hydroxytryptophan as a new intrinsic probe for investigating protein-DNA interactions by analytical ultracentrifugation. Study of the effect of DNA on self-assembly of the bacteriophage lambda cI repressor.

Pairwise cooperativity between proteins bound to DNA is believed to be important in governing the transcriptional regulation of numerous genes. However, the spectral overlap of normal proteins and DNA has blocked the study of these interactions by many physical methods. As shown recently by Ross et al. (in press), lambda cI repressor spectrally enhanced by 5-hydroxytryptophan (5-OHTrp), expressed in vivo using an Escherichia coli tryptophan auxotroph, exhibits dimer formation and DNA binding properties identical with those of the wild-type repressor. Moreover, the 5-OHTrp provides a spectral signal that allows monitoring of the protein concentration without interference from DNA. In this article, the ability to selectively detect 5-OHTrp-labeled repressor during analytical ultracentrifugation is used to study the higher order assembly of repressor dimers in the absence and in the presence of operator DNA. Contrary to the expectation that tetramer might be the limiting oligomer, lambda cI repressor undergoes a definite association to octamer. The relatively narrow concentration range over which transition from predominantly dimer to predominantly octamer occurs makes it unlikely that significant levels of tetramer are formed in the absence of DNA. Moreover, mass measurements reveal that an OR1 oligonucleotide binds to octameric repressor and does not dissociate it to tetramers. The use of the 5-OHTrp spectral enhancement opens a promising new avenue for the exploration of protein-protein and protein-nucleic acid interactions by analytical ultracentrifugation.

Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.

Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay.

We have investigated the question of whether the gel mobility-shift assay can provide data that are useful to the demonstration of cooperativity in the site-specific binding of proteins to DNA. Three common patterns of protein-DNA interaction were considered: (i) the cooperative binding of a protein to two sites (illustrated by the Escherichia coli Gal repressor); (ii) the cooperative binding of a bidentate protein to two sites (illustrated by the E. coli Lac repressor); and (iii) the cooperative binding of a protein to three sites (illustrated by the lambda cI repressor). A simple, rigorous, and easily extendable statistical mechanical approach to the derivation of the binding equations for the different patterns is presented. Both simulated and experimental data for each case are analyzed. The mobility-shift assay provides estimates of the macroscopic binding constants for each step of ligation based on its separation of liganded species by the number of ligands bound. Resolution of the binding constants depends on the precision with which the equilibrium distribution of liganded species is determined over the entire range of titration of each of the sites. However, the evaluation of cooperativity from the macroscopic binding constants is meaningful only for data that are also accurate. Some criteria that are useful in evaluating accuracy are introduced and illustrated. Resolution of cooperative effects is robust only for the simplest case, in which there are two identical protein binding sites. In this case, cooperative effects of up to 1,000-fold are precisely determined. For heterogeneous sites, cooperative effects of greater than 1,000-fold are resolvable, but weak cooperativity is masked by the heterogeneity. For three-site systems, only averaged pair-wise cooperative effects are resolvable.

Comparison of operator-specific and nonspecific DNA binding of the lambda cI repressor: [KCl] and pH effects.

The effects of proton and KCl activity on the nonspecific lambda cI repressor-DNA interactions and on the site-specific repressor-O(R) interactions were compared, in order to assess their roles in site specificity. The repressor-O(R) interactions were studied by using DNase I footprint titration. The Gibbs free energy changes for binding and for cooperativity were determined between 25 and 300 mM KCl, from individual-site isotherms for the binding of repressor to O(R) and to reduced-valency mutants. The proton-linked effects on repressor-O(R) interactions have been published [Senear, D. F., & Ackers, G. K. (1990) Biochemistry 29, 6568-6577; Senear, D. F., & Bolen, D. W. (1991) Methods Enzymol. (in press)]. Nonspecific binding was studied by using a nitrocellulose filter binding assay, which proved advantageous in this case, due to the relatively weak nonspecific binding, and precipitation of repressor-DNA complexes. Filter binding provided measurements at low binding density where precipitation did not occur. The data provide estimates of the Gibbs free energy changes for nonspecific, intrinsic binding, but not for cooperativity. The KCl concentration dependencies of the intrinsic binding constants indicate that ion release plays similar roles in distinguishing between the operators and in discriminating operator from nonoperator DNA. Binding to DNA is accompanied by net proton absorption. Near neutral pH, proton linkages to operator and nonoperator binding are the same. Differences at acid and at basic pH implicate the same ionizable repressor groups in distinguishing between the operators and in discriminating operator from nonoperator DNA. The results indicate similar overall modes of operator and nonoperator binding of repressor, but implicate indirect effects of DNA sequence as important contributors to sequence recognition.

Proton-linked contributions to site-specific interactions of lambda cI repressor and OR.

The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.

Flanking DNA-sequences contribute to the specific binding of cI-repressor and OR1.

The binding of cI-repressor to a series of mutant operators containing OR1 of the right operator of bacteriophage lambda was investigated. Sites OR2 and/or OR3 were inactivated by either point or deletion mutations. The free energy of binding repressor to OR1 in the wildtype operator, delta G1, is -13.7 +/- 0.3 kcal/mol. delta G1 determined for an OR2- operator created by a single point mutation in OR2 is -13.6 +/- 0.2 kcal/mol. In contrast, delta G1 for the binding of repressor to a cloned synthetic OR1 operator containing only 24 bp of lambda sequence is -12.2 +/- 0.1 kcal/mol. When sequence 5' to OR1 is present, the binding affinity increases to -13.0 +/- 0.1 kcal/mol. In addition, the proximity of OR1 to a fragment-end decreases delta G1 from -13.7 to -12.3 +/- 0.1 kcal/mol. These results suggest that the DNA sequence outside the 17 bp OR1 binding-site contributes to the specific binding of cI-repressor.

Thermodynamic investigation of monoclonal antibody alkylated nucleoside interaction as a model for epitope recognition on nucleic acids.

The objective of this investigation is examination of the dominant forces that govern complex formation between a series of monoclonal antibodies directed against O6-ethyl-2'-deoxyguanosine. These monoclonal antibodies (coded as ER-6, ER-3, and EM-1) provide the basis for a thermodynamic comparative evaluation of the potentially different forces that stabilize the various monoclonal antibody (mAb) alkylated nucleoside complexes. The binding affinities of ER-6, ER-3, and EM-1 are measured in terms of specific (O6-ethyl-2'-deoxyguanosine, or O6-EtdGuo) and nonspecific (O6-methyl-2'-deoxyguanosine, or O6-MedGuo) antigens, under a variety of experimental conditions, including pH, sodium chloride addition, 1-propanol addition, and temperature, via a nitrocellulose affinity filter assay. The binding isotherms were analyzed via a least-squares routine fit to a two independent binding sites model. The temperature dependence of the van't Hoff enthalpies for the specific O6-EtdGuo interaction ranges from -15.18 to -18.60 kcal mol-1, while for O6-MedGuo the range was extended from -2.72 to -20.66 kcal mol-1. The standard and unitary entropies were negative for those mAb interactions with O6-EtdGuo as well as for ER-6/O6-MedGuo complex formation. However, it was found that the interactions between ER-3 and EM-1 with O6-MedGuo led to decidedly positive entropic values. These results indicate two different dominant forces at work in complex stabilization. The interaction of the three mAb's with their specific antigen, as well as ER-6/O6-MedGuo interaction (nonspecific), may well be controlled by van der Waals type forces, while ER-3 and EM-1 interactions with nonspecific antigen imply formal charge neutralization electrostatics as the dominant force.