Stable, monomeric variants of lambda Cro obtained by insertion of a designed beta-hairpin sequence.

lambda Cro is a dimeric DNA binding protein. Random mutagenesis and a selection for Cro activity have been used to identify the contacts between Cro subunits that are crucial for maintenance of a stably folded structure. To obtain equivalent contacts in a monomeric system, a Cro variant was designed and constructed in which the antiparallel beta-ribbon that forms the dimer interface was replaced by a beta-hairpin. The engineered monomer has a folded structure similar to wild type, is significantly more stable than wild type, and exhibits novel half-operator binding activity.

Upstream operators enhance repression of the lac promoter.

To study regulation of transcription by distant elements, a wild-type lac operator was inserted upstream of a promoter-constitutive operator control region. The upstream operator is shown to aid in repression of transcription from the mutant control region. The effectiveness of the upstream operator as a function of its distance from the mutant control region parallels the length dependence observed for DNA cyclization. A quantitative model is proposed for action-at-a-distance of DNA control sites in which protein-protein and protein-DNA interactions are mediated by DNA looping. In this model, the effective concentrations of interacting proteins that are tethered by DNA are determined by the length of the intervening DNA and by its inherent bending and torsional stiffness. This model makes a number of predictions for both eukaryotic and prokaryotic control sequences located far from their sites of action.

Hamiltonians for protein tertiary structure prediction based on three-dimensional environment principles.

We describe a computational approach to protein tertiary structure prediction that combines ideas from the three-dimensional (3D) profile method of Bowie, Lüthy and Eisenberg and the associative memory Hamiltonians of Friedrichs and Wolynes. The ultimate goal of our work is to extend and generalize the capabilities of these heuristics so as to be able to predict novel structures that might be found in nature or designed proteins. In our approach we approximate the interactions between residues through a pseudo-potential function similar to an associative memory Hamiltonian. This function is constructed based on 3D environment principles. Favorable inter-residue contacts for each residue in a target protein are inferred by using 3D environment propensities of the residues and a collection of 3D environment templates derived from a dataset of protein crystal structures. A Hamiltonian encoding this information is used to guide an optimization phase via molecular dynamics with annealing, which then leads to the folded structure. With our algorithm we can recover the structure of dataset proteins and have also succeeded in constructing the fold for a protein with little sequence similarity to any dataset protein.

Thermodynamic origins of specificity in the lac repressor-operator interaction. Adaptability in the recognition of mutant operator sites.

A system has been developed for facile generation and characterization of mutant lac operator sites, free of competing pseudo operator sequences. The interaction of lac repressor with these sites has been investigated by the nitrocellulose filter binding assay. The equilibrium binding affinity for each of three single-site changes was reduced by more than three orders of magnitude relative to the wild-type operator under standard assay conditions. The free-energy changes associated with single base-pair substitutions are not additive. We propose that adaptations in the recognition surface of the repressor involving significant trade-offs between electrostatic versus non-electrostatic interactions and between enthalpic versus entropic contributions to the binding free energy occur, in order to achieve the most stable complex with a given DNA sequence.

A folded monomeric intermediate in the formation of lambda Cro dimer-DNA complexes.

The folding, dimerization and DNA binding equilibria of the bacteriophage lambda Cro repressor have been characterized. Comparison with four engineered variants shows that a folded monomeric species is substantially populated under conditions used for the formation of dimer-DNA complexes. Although Cro dimers are the only DNA-bound species observed in electrophoretic mobility shift assays, cooperativity in Cro-DNA binding isotherms shows that the predominant free protein species is monomeric at nanomolar concentrations. Micromolar dissociation constants for Cro dimers have been measured in the absence of DNA by sedimentation equilibrium and gel filtration chromatography. Denaturation of Cro dimers in the 10 to 100 micromolar concentration range by guanidine hydrochloride (GdnHCl) is well modeled as a two-state process, with folded dimers and unfolded monomers as the only significantly populated species. However, linear extrapolation of this composite unfolding and dimer dissociation free energy predicts a nanomolar dissociation constant in the absence of denaturant. This extrapolation is clearly inconsistent with the DNA binding and hydrodynamic measurements. Our interpretation of these results is that the monomeric species detected in DNA binding and hydrodynamic experiments is predominantly folded. The stability of the folded monomeric species can be calculated as the difference between the dimerization free energy determined from hydrodynamic measurements and the folding free energy extrapolated from GdnHCl denaturation. The calculated stability of the Cro F58W monomer is greater than that of the wild-type Cro monomer. Thus, residue 58, which makes critical intermolecular contacts across the dimer interface, is also involved in intramolecular stabilization of the monomeric intermediate.

The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers.

It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity. The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.

Crystal structure of an engineered Cro monomer bound nonspecifically to DNA: possible implications for nonspecific binding by the wild-type protein.

The structure has been determined at 3.0 A resolution of a complex of engineered monomeric Cro repressor with a seven-base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half-operator that is recognized by each subunit of the wild-type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence-independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48 degrees further out of the major groove, while the turn region of the helix-turn-helix remains in contact with the DNA backbone. All of the direct base-specific interactions seen in the wild-type Cro-operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein DNA interface than for half of the wild-type Cro-operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence-nonspecific interaction of the wild-type Cro dimer with DNA. In the sequence-specific complex, the DNA is bent, the protein dimer undergoes a large hinge-bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.

Physical properties of DNA in vivo as probed by the length dependence of the lac operator looping process.

Plasmid constructs containing a wild-type (O+) lac operator upstream of an operator-constitutive (Oc) lac control element exhibit a length-dependent, oscillatory pattern of repression of expression of the regulated gene as interoperator spacing is varied from 115 to 177 base pairs (bp). Both the length dependence and the periodicity of repression are consistent with a thermodynamic model involving a stable looped complex in which bidentate lac repressor interacts simultaneously with both O+ and Oc operators. The oscillatory pattern of repression with distance occurs with a period approximating the helical repeat of DNA and presumably reflects the necessity for proper alignment of interacting operators along the helical face of the DNA. In the length regime examined, the presence of the upstream operator enhances repression between 6-fold and 50-fold depending upon phasing. This reflects a torsional rigidity of DNA in vivo that is consistent with in vitro measurements. The oscillatory pattern of repression is best fit with a period of either 9.0 or 11.7 bp/cycle but not 10.5 bp/cycle. This periodicity is interpreted as reflecting the average helical repeat of the 40-bp interoperator region of plasmid DNA in vivo, suggesting that the local helical repeat of DNA in vivo may differ significantly from 10.5 bp/turn. The apparent persistence length needed to fit the data (aapp) is only one-fifth the standard in vitro value. This low value of aapp may be due in part to DNA bending induced by catabolite activator protein (CAP) bound to its site between the interacting operators.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific recognition by an isolated DNA-binding domain of the sine oculis protein.

The sine oculis (so) gene is required for the development of the Drosophila visual system. The 416 amino acid SO protein contains a 40 amino acid region homologous to the helix-turn-helix (HtH) region of the homeodomain. Three HtH-containing peptides ranging in size from 63 to 93 amino acids (SO(218-279), SO(204-279), and SO(188-279)) were expressed in Escherichia coli and characterized in vitro. These fragments show circular dichroism spectra characteristic of helical proteins and cooperative unfolding transitions. Derivatization of these three peptides with the chemical nuclease 1,10-phenanthroline:copper (OP-Cu) allowed the identification of specific DNA-binding sites within the 3.1 kb pUC119 plasmid. Similar cleavage patterns with similar relative affinities were obtained for all three peptides. Nucleotide resolution mapping of the predominant cleavage area identified two primary cleavage sites with a similar core sequence. The DNA cleavage sites were confirmed by DNase I footprinting with both native and OP-Cu-conjugated SO HtH peptides. This study identifies a 63 amino acid peptide as sufficient for specific DNA binding.

Functional expression and affinity selection of single-chain cro by phage display: isolation of novel DNA-binding proteins.

A robust selection system affording phage display of the DNA-binding helix-turn-helix protein Cro is presented. The aim of the work was to construct an experimental system allowing for the construction and isolation of Cro-derived protein with new DNA-binding properties. A derivative of the phage lambda Cro repressor, scCro8, in which the protein subunits had been covalently connected via a peptide linker was expressed in fusion with the gene 3 protein of Escherichia coli filamentous phage. The phage-displayed single-chain Cro was shown to retain the DNA binding properties of its wild-type Cro counterpart regarding DNA sequence specificity and binding affinity. A kinetic analysis revealed the rate constant of dissociation of the single-chain Cro-phage/DNA complex to be indistinguishable from that of the free single-chain Cro. Affinity selection using a biotinylated DNA with a target consensus operator sequence allowed for a 3000-fold enrichment of phages displaying single-chain Cro over control phages. The selection was based on entrapment of phage/DNA complexes formed in solution on streptavidin-coated paramagnetic beads. The expression system was subsequently used to isolate variant scCro8 proteins, mutated in their DNA-binding residues, that specifically recognized new, unnatural target DNA ligands.

High-resolution structure of an engineered Cro monomer shows changes in conformation relative to the native dimer.

A rationally designed, genetically engineered, monomeric form of the Cro protein from bacteriophage lambda has been crystallized and its structure determined by isomorphous replacement and refined to a resolution of 1.54 A. The structure confirms the rationale of the design but, at the same time, reveals 1-2 A shifts throughout the monomer structure relative to the previously determined structure of the dimeric wild-type protein. These changes include a 1.6 A main-chain shift in part of the beta-sheet region of the molecule relative to the alpha-helical region and a 1.1 A shift of a buried phenylalanine within the core as well as a correlated 2.2 A shift in a solvent-exposed beta-hairpin. The conformational adjustments appear to reflect an inherent flexibility of the protein that is associated with its DNA-binding function.

Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis.

The crystal structure of an engineered monomer of the lambda Cro repressor shows unexpected expansion of the hydrophobic core of the protein and disorder of the five C-terminal residues [Albright et al. (1996) Biochemistry 35, 735-742]. This structural information has guided the construction of a second generation of monomeric Cro proteins by combinatorial mutagenesis of selected core and C-terminal residues. Clones were identified in a library of randomized cro genes by a genetic screen for protein accumulation in Escherichia coli. Sequencing of candidate genes followed by purification and analysis of their product proteins has identified alternative arrangements of hydrophobic core residues which result in substantial increases in thermal stability. In contrast, residue replacements at the C-terminus have minor effects on stability but may increase protein expression levels.

Single-chain lambda Cro repressors confirm high intrinsic dimer-DNA affinity.

The overall affinity of the bacteriophage lambda Cro repressor for its operator DNA site is limited by dimer dissociation at submicromolar concentrations. Since Cro dimer-operator complexes form at nanomolar concentrations of Cro subunits where free dimers are rare, these dimers must bind with compensating high affinities. Previous studies of the covalent dimer Cro V55C suggest little change in DNA binding affinity even though the dimeric species is quantitatively populated; this is an apparent contradiction to the expectation of high intrinsic dimer-DNA affinity. In contrast to the disulfide linkage at the center of the dimer interface in Cro V55C, polypeptide linkers that join the two subunits allow single-chain Cro repressors to bind operator DNA with picomolar affinities. A series of five single-chain Cro repressors have been expressed from fused tandem cro genes. Each contains a peptide linker of 8-16 hydrophilic residues that connects the C-terminus of one subunit to the N-terminus of the next. All bind to operator DNA with at least 100-fold higher affinity than Cro V55C. Proteins containing the longest and shortest linkers have been purified and characterized in detail. Both exhibit similar CD spectra to wild-type Cro and enhanced thermal stability. Sedimentation equilibrium experiments show that single-chain Cro repressors do not associate at concentrations up to 30 microM. The rate of dissociation of Cro-DNA complexes is almost unchanged by covalent linkage. Biophysical characterization of Cro variants such as these, where DNA binding is uncoupled from subunit assembly, is necessary for a quantitative understanding of the structural and energetic determinants of DNA recognition in this simple model system.

Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression.

Effects of changes in intracellular ion concentrations on the interactions of Escherichia coli lac repressor with lac operator mutants and on the interactions of RNA polymerase with various promoters have been investigated in vivo. The intracellular ionic environment was reproducibly varied by changing the osmolality of the 4-morpholinepropanesulfonic acid minimal growth medium. As the osmolality of the growth medium is varied from 0.1 to 1.1 osmolal, the total intracellular concentration of K+ increases linearly from 0.23 +/- 0.03 to 0.93 +/- 0.05 molal and the total intracellular concentration of glutamate increases linearly from 0.03 +/- 0.01 to 0.26 +/- 0.02 molal. The sum of the changes in the total concentrations of these two ions appears sufficient to compensate for a given change in external osmolality, indicating that K+ and glutamate are the primary ionic osmolytes under these conditions and that these ions are free in the cytoplasm. In support of this, in vivo 39K NMR experiments as a function of external osmolality indicate that changes in the total cytoplasmic K+ concentration correspond to changes in the free cytoplasmic K+ concentration. Extents of interaction of lac repressor and RNA polymerase with their specific DNA sites were monitored by measuring the amounts of beta-galactosidase produced under the control of these sites. For both lac repressor and RNA polymerase, it was found that formation of functional protein-DNA complexes in vivo is only weakly (if at all) dependent on intracellular ion concentration. These results contrast strongly with those obtained on these systems in vitro, which showed that both the equilibria and kinetics of binding are extremely salt-dependent. We discuss several possible mechanisms by which E. coli may compensate for the potentially disruptive effects of these large changes in the intracellular ionic environment.