Biomedical subjects

R T Sauer

Publications and source records attributed to R T Sauer.

At least 145 records · Page 8Linked to original sources

PubMed1987

1H NMR aromatic spectrum of the operator binding domain of the lambda repressor: resonance assignment with application to structure and dynamics.

The aromatic 1H NMR resonances of the operator binding domain of lambda repressor are completely assigned. Since the resonances of this 23-kilodalton domain are too broad for the application of two-dimensional strategies for sequence-specific assignment, an alternative approach has been used. Assignments are obtained by a combination of one- and two-dimensional NMR methods, by the study of genetically altered domains, and by the biosynthetic incorporation of deuterium labels. The resulting assignments provide sensitive markers for tertiary and quaternary structure. Nuclear Overhauser enhancements demonstrate that the major features of the crystal structure, including the dimer contacts, are retained in solution. The rates of aromatic ring rotation indicate that the globular domain is not rigid; significant barriers to ring rotation are observed only in the dimer contact.

Bacteriophage lambda↗

PubMed1987

Dimerization of the operator binding domain of phage lambda repressor.

Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.

Amino Acids↗

PubMed1987

Quaternary structure and function in phage lambda repressor: 1H-NMR studies of genetically altered proteins.

The quaternary structure and dynamics of phage lambda repressor are investigated in solution by 1H-NMR methods. lambda repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. 1H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the "linker" polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84----Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.

Bacteriophage lambda↗

PubMed1986

Interaction of mutant lambda repressors with operator and non-operator DNA.

We have described a set of mutations that alter side-chains on the operator binding surface of lambda repressor. In this paper, we study the interactions of 12 purified mutant repressors with operator and non-operator DNA. The mutant proteins have operator affinities that are reduced from tenfold to greater than 10,000-fold compared to wild-type. Nine of the mutants have affinities for non-operator DNA that are similar to wild-type, two mutants show decreased non-specific binding, and one mutant has increased affinity for non-operator DNA. We discuss these findings in terms of the structural and energetic contributions of side-chain--DNA interactions, and show that certain contacts between the repressor and the operator backbone contribute both energy and specificity to the interaction.

Amino Acid Sequence↗

PubMed1986

Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions.

Under physiological conditions, lambda repressor can be inactivated in vivo or in vitro by RecA-mediated cleavage of the polypeptide chain. The repressor protein is thought to cleave itself, with RecA acting to stimulate autodigestion. ind- repressor mutants are resistant to RecA-mediated inactivation in vivo. In this paper, we report the purification of 15 ind- repressor proteins and the behaviors of these proteins in the RecA-mediated and autodigestion cleavage reactions. None of these proteins undergoes substantial RecA-dependent cleavage. However, eight mutant proteins autodigest at the same rate as wild-type repressor, six mutants do not autodigest or autodigest slower, and one mutant autodigests faster than wild-type. We discuss these results with respect to repressor structure and RecA-binding, and suggest possible roles for the RecA protein in the cleavage reaction.

DNA, Viral↗

PubMed1986

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor.

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.

Amino Acid Sequence↗

PubMed1986

Bacteriophage P22 Cro protein: sequence, purification, and properties.

The DNA sequence of part of the bacteriophage P22 early regulatory region, including genes cro and c1, was determined. The protein product of the cro gene consists of 61 amino acid residues, and that of c1, 92 amino acid residues. Both genes were placed separately in plasmids from which they are expressed from a controllable promoter in vivo. Induced cells bearing the cro-expressing plasmid were used as a source for purifying and characterizing the Cro protein. The amino-terminal sequence of this protein was found to be as predicted by the DNA sequence; close agreement was also observed between its predicted and experimentally determined amino acid composition and molar extinction coefficient at 280 nm. In gel filtration experiments, Cro protein at concentrations around 10(-5) M appears to have a molecular weight of 8600, which is more consistent with monomers (6800) than with dimers (13 600). Cro protein binds specifically to the three repressor binding sites in the P22 right operator; in order of decreasing affinity, these are OR3, OR1, and OR2.

Amino Acid Sequence↗

PubMed1986

Stabilization of lambda repressor against thermal denaturation by site-directed Gly----Ala changes in alpha-helix 3.

Oligonucleotide-directed mutagenesis has been used to replace alpha-helical glycines in the N-terminal domain of lambda repressor with alanines. Since alanine is a significantly better helix-forming residue than glycine, these changes were predicted to have a stabilizing effect. We show that the Gly46----Ala substitution, the Gly48----Ala substitution, and the double substitution increase the melting temperature of the N-terminal domain by 3-6 degrees.

Amino Acid Sequence↗

PubMed1986

Isolation and analysis of arc repressor mutants: evidence for an unusual mechanism of DNA binding.

We have isolated 64 different missense mutations at 36 out of 53 residue positions in the Arc repressor of bacteriophage P22. Many of the mutant proteins with substitutions in the C-terminal 40 residues of Arc have reduced intracellular levels and probably have altered structures or stabilities. Mutations in the N-terminal ten residues of Arc cause large decreases in operator DNA binding affinity without affecting the ability of Arc to fold into a stable three-dimensional structure. We argue that these N-terminal residues are important for operator recognition but that they are not part of a conventional helix-turn-helix DNA binding structure. These results suggest that Arc may use a new mechanism for sequence specific DNA binding.

Amino Acid Sequence↗

PubMed1986

Bacteriophage lambda cro mutations: effects on activity and intracellular degradation.

Following random mutagenesis of the bacteriophage lambda cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type.

Amino Acid Sequence↗

PubMed1985

Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins.

We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.

Amino Acid Sequence↗

PubMed1985

The bacteriophage P22 arc and mnt repressors. Overproduction, purification, and properties.

The arc and mnt genes of bacteriophage P22 encode small repressor proteins. We have cloned these genes onto plasmids that overproduce Arc and Mnt to greater than 1% of the soluble cellular protein. Both proteins were purified to greater than 95% homogeneity, and N-terminal sequences and amino acid compositions were determined. These data, in combination with previously determined gene sequences, establish the complete protein sequences for Arc (53 residues) and Mnt (82 residues). Both proteins have melting temperatures between 45 and 55 degrees C and can be renatured to a fully active species. Arc is a dimer in solution and Mnt is a tetramer.

Amino Acid Sequence↗

PubMed1985

Crystallization of the Arc repressor.

Arc, a repressor from Salmonella phage P22 has been crystallized from ammonium phosphate at pH 8.0. The crystals form in space group P212121 with a = 90.26 A, b = 52.88 A and c = 47.58 A. The crystals diffract to 2.2 A resolution.

Crystallography↗

PubMed1985

Increasing and decreasing protein stability: effects of revertant substitutions on the thermal denaturation of phage lambda repressor.

The thermal denaturations of five revertant lambda repressors containing single amino acid substitutions in their N-terminal domains have been studied by differential scanning calorimetry. Two substitutions slightly decrease stability, and the remaining three render the protein more stable than wild type. The Gly48----Asn and Gly48----Ser proteins are 4 degrees C more stable than wild type. These two substitutions replace an alpha helical residue, and in each case a poor helix forming residue, glycine, is replaced by a residue with a higher helical propensity. We also present data showing that one revertant, Tyr22----Phe, has reduced operator DNA binding affinity despite its enhanced stability.

Amino Acid Sequence↗

PubMed1985

Lambda repressor mutations that increase the affinity and specificity of operator binding.

Intragenic, second-site reversion has been used to identify amino acid substitutions that increase the affinity and specificity of the binding of lambda repressor to its operator sites. Purified repressors bearing the second-site substitutions bind operator DNA from 3 to 600 fold more strongly than wild type; these affinity changes result from both increased rates of operator association and decreased rates of operator dissociation. Three of the revertant substitutions occur in the alpha 2 and alpha 3 DNA binding helices of repressor and seem to increase affinity by introducing new salt-bridges or hydrogen bonds with the sugar-phosphate backbone of the operator site. The fourth substitution alters the alpha 5 dimerization helix of repressor and appears to increase operator affinity indirectly.

Amino Acid Sequence↗

PubMed1985

Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage.

In this paper, we report on the isolation and sequence analysis of mutations that confer an induction-deficient phenotype to lambda repressor. A total of 16 different mutations, which occur at 13 different sites in the repressor gene, have been characterized. For most of the mutant lysogens, frequencies of spontaneous induction in a recA+ strain were reduced dramatically in comparison with those for a wild-type phage, and these mutant lysogens showed little or no prophage induction after UV irradiation. The immunity properties of cells containing the mutant repressors show that all of the mutants but one exhibit operator-binding properties indistinguishable from wild-type repressor.

Bacteriophage lambda↗

PubMed1984

Amino-terminal arm of the lambda repressor: a 1H NMR study.

The N-terminal arm of the lambda repressor is shown to be flexible in solution by one- and two-dimensional 1H NMR methods. In particular, the relaxation of Thr-2 is largely independent of macromolecular tumbling. The conformation of the operator-binding domain is not affected by the removal of the first three residues nor by a point mutation, Lys-4----Gln. These results support a proposed model of the lambda repressor-operator complex in which the N-terminal arm of the repressor is assumed to be flexible and to wrap around the operator double helix.

DNA-Binding Proteins↗

PubMed1984

Structure of tomato bushy stunt virus. V. Coat protein sequence determination and its structural implications.

We report the chemically determined sequence of most of the polypeptide chain of the coat protein of tomato bushy stunt virus. Peptide locations have been determined by comparison with the high-resolution electron density map from X-ray crystallographic analysis as well as by conventional chemical overlaps. Three small gaps remain in the 387-residue sequence. Positively charged side-chains are concentrated in the N-terminal part of the polypeptide (the R domain) as well as on inward-facing surfaces of the S domain. There is homology of S-domain sequences with structurally corresponding residues in southern bean mosaic virus.

Amino Acid Sequence↗