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R T Sauer

Publications and source records attributed to R T Sauer.

At least 127 records · Page 7Linked to original sources
PubMed1989

Production and characterization of monoclonal antibodies to the N-terminal domain of lambda repressor.

Monoclonal antibodies reactive with distinct regions of the N-terminal domain of the lambda repressor protein have been isolated. By comparing the affinities of these antibodies for mutant repressors with increased and decreased thermal stabilities, each of the antibodies can be shown to bind to epitopes accessible in the native conformation of the N-terminal domain. Experiments probing antibody binding to protein fragments, mutant variants, and peptides have also been used to define likely regions of contact between the antibodies and the N-terminal domain.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of the fine specificity of binding of monoclonal antibody 51F to lambda repressor.

Monoclonal antibody 51F recognizes determinants in the helix 4 region of the native form of the N-terminal domain of lambda repressor. A cassette mutagenesis method was used to introduce changes within this region, and antibody-reactive candidates were isolated and sequenced. The resulting data allow the identification of repressor side chains that are critical determinants of antibody binding. Four of these side chains are on the surface of the N-terminal domain and probably contact the antibody directly. These contact positions were then mutagenized individually, and the antibody binding phenotypes of a large number of singly mutant repressors were determined. Taken together, the mutational data allow a functional map of the recognition surface to be constructed and the physical nature of some of the specific interactions that stabilize the antibody-antigen complex to be surmised.

Amino Acid Sequence↗
PubMed1989

The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli.

To investigate the relationship between the degradation rate of a protein in Escherichia coli and its thermal stability in vitro, we constructed a set of variants of the N-terminal domain of lambda repressor with a wide range of melting temperatures. Pulse-chase experiments showed that, within this set, the proteins that are most thermally stable have the longest intracellular half-lives and vice versa. Moreover, second-site mutations which act directly or indirectly to increase the thermodynamic stability of the native N-terminal domain were found to suppress the intracellular degradation of one of the unstable mutants. These data suggest that thermal stability is, indeed, a key determinant of the proteolytic susceptibility of this protein in the cell. It is not the sole determinant, however, as sequences at the extreme C terminus of the N-terminal domain can influence proteolytic sensitivity without affecting the stability of the native structure. We propose that the thermal stability of the N-terminal domain of lambda repressor is an important determinant of its proteolytic sensitivity because degradation proceeds primarily from the unfolded form and that sequence determinants within the unfolded chain influence whether the unfolded protein will be a good substrate for proteolytic enzymes.

Bacterial Proteins↗
PubMed1989

Identification of C-terminal extensions that protect proteins from intracellular proteolysis.

Revertants of defective mutants in the Arc repressor of bacteriophage P22 were isolated. Five of the six reverting mutations were frameshifts near the end of the coding sequence which resulted in proteins with C-terminal extensions. Each of the reverting mutations prolong the half-lives in vivo of the proteins in which they reside, yet they do not alter the thermodynamic stability, structure, oligomeric form, or DNA-binding properties of these proteins. Fusion of one of these tails to the C-terminal end of a mutant form of the N-terminal domain of lambda repressor also prevented proteolysis of this protein. These C-terminal sequences may prevent degradation by blocking the recognition of unstable proteins by the proteolytic machinery in the cell.

Amino Acid Sequence↗
PubMed1989

Alternative packing arrangements in the hydrophobic core of lambda repressor.

The random alteration of hydrophobic core positions in the N-terminal domain of lambda-repressor, both individually and in combination, shows that there are many ways of repacking the core of the protein. Although the number of functional sequences is limited by constraints on composition, volume and steric interactions, the simple requirement that these positions remain hydrophobic is the main determinant of whether a core sequence is compatible with the wild-type fold.

Amino Acid Sequence↗
PubMed1989

Lambda repressor mutants that are better substrates for RecA-mediated cleavage.

RecA-mediated cleavage of the bacteriophage lambda repressor results in inactivation of the protein and leads to induction of the lambda prophage. Here, we report the identification of three mutations in lambda repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the ind- mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.

Bacteriophage lambda↗
PubMed1989

The Arc and Mnt repressors. A new class of sequence-specific DNA-binding protein.

Genetic, biochemical, and biophysical studies have begun to reveal details of the structures of Arc and Mnt and show that these repressors use residues at their N-terminal ends for operator recognition and binding. Some of the DNA contacts made by these residues have been identified, and this information together with NMR studies has permitted the construction of models of the DNA binding region. Although the accuracy of these models remains to be determined, it seems clear that Arc and Mnt are members of a new class of DNA-binding proteins.

Amino Acid Sequence↗
PubMed1989

Sequence-specific binding of arc repressor to DNA. Effects of operator mutations and modifications.

A set of arc operators with transition and/or transversion mutations at each operator base pair has been constructed. By determining the ability of Arc to bind these variant operators, the importance of each base pair for Arc recognition has been assessed. Methylation protection experiments have also been used to probe points of close contact between Arc and most of the mutant operators. These data, together with phosphate interference results obtained previously for the wild type operator, provide information about the operator surface that is contacted when Arc binds.

Bacteriophages↗
PubMed1989

Amino acid substitutions that increase the thermal stability of the lambda Cro protein.

A mutant Cro protein, which bears the Ile-30----Leu substitution, is thermally unstable and degraded more rapidly than wild-type Cro in vivo. Using an antibody screen, we have isolated five different second site suppressor substitutions that reduce the proteolytic hypersensitivity of this mutant Cro protein. Two of the suppressor substitutions increase the thermal stability of Cro by 12 degrees C to 14 degrees C. These amino acid substitutions affect residues 16 and 26, which are substantially exposed to solvent in the crystal structure of wild-type Cro.

Amino Acid Sequence↗
PubMed1989

Identifying determinants of folding and activity for a protein of unknown structure.

We have generated an extensive genetic map of functionally allowed and/or structurally allowed amino acid substitutions in Arc repressor, a DNA binding protein of unknown structure. Analysis of the allowed substitution patterns identifies residues that are likely to be involved in protein function and identifies side chains that play important structural roles, including residues likely to form the hydrophobic core. The identities of approximately one-third of the residues in Arc repressor are functionally important, about one-half are structurally important, and the remainder are unimportant for either structure or function. The patterns of obligatory hydrophobic positions permit strong predictions of secondary structure.

Amino Acid Sequence↗
PubMed1989

Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation.

To test the idea that unfolded protein might act as an intracellular signal for induction of the heat shock response in Escherichia coli, we examined the synthesis of several heat shock proteins after expression of an unfolded variant of the amino-terminal domain of lambda repressor. These experiments show that expression of a single mutant protein, and not its wild-type counterpart, is sufficient to induce a heat shock-like response. In addition, by measuring the abilities of unfolded variants of differing proteolytic susceptibilities to induce heat shock protein synthesis and by monitoring heat shock protein synthesis as a function of the amount of a single unfolded protein, we show that it is the concentration of unfolded protein in the cell, and not its degradation, that is important for inducing the heat shock-like response.

Bacterial Proteins↗
PubMed1989

Genetic analysis of protein stability and function.

There is tremendous variability in the importance of individual amino acids in protein sequences. On the one hand, nonconservative residue substitutions can be tolerated with no loss of activity at many residue positions, especially those exposed on the protein surface. On the other hand, destabilizing mutations can occur at a large number of different sites in a protein, and for many proteins such mutations account for more than half of the randomly isolated missense mutations that confer a defective phenotype. At sites that are key determinants of stability or activity, even residue substitutions that are generally considered to be conservative (e.g., Glu in equilibrium Asp, Asn in equilibrium Asp, Ile in equilibrium Leu, Lys in equilibrium Arg and Ala in equilibrium Gly) can have severe phenotypic effects. Unfortunately, this means that there is no simple way to infer the likely effect of an amino acid substitution on the basis of sequence information alone. A nonconservative Gly----Arg substitution could be phenotypically silent at one position while a conservative Asn----Asp change could lead to complete loss of activity at another position. For proteins whose structures are known, it is often possible to predict whether particular residue substitutions will be destabilizing, as long as detailed estimates of the destabilization energy are not required. Substitutions that introduce polar groups, large cavities, or overly large side chains into the hydrophobic core are potentially the most destabilizing. Substitutions that disrupt hydrogen bonding or electrostatic interactions can also have significant effects, although the destabilization caused by these substitutions is smaller than that caused by severe core mutations. Destabilizing substitutions that involve replacing glycines in turns, or introducing prolines into alpha-helices and other disallowed positions are also reasonably common. Finally, most solvent exposed residues can apparently be freely substituted without serious effects on protein stability. Although exceptions may occur, these generalizations serve to summarize a large body of information and can be rationalized in physical and chemical terms. It is an especially encouraging result that proteins appear to tolerate most substitutions, even those that are destabilizing, without significant changes in the native structure. For proteins whose structures are known, this means that it is reasonable to interpret mutant phenotypes in terms of the wild-type structure. For proteins whose structures are not known, it is reasonable to infer that mutations that reduce activity without affecting stability are directly involved in function.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1988

Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences.

A method of combinatorial cassette mutagenesis was designed to readily determine the informational content of individual residues in protein sequences. The technique consists of simultaneously randomizing two or three positions by oligonucleotide cassette mutagenesis, selecting for functional protein, and then sequencing to determine the spectrum of allowable substitutions at each position. Repeated application of this method to the dimer interface of the DNA-binding domain of lambda repressor reveals that the number and type of substitutions allowed at each position are extremely variable. At some positions only one or two residues are functionally acceptable; at other positions a wide range of residues and residue types are tolerated. The number of substitutions allowed at each position roughly correlates with the solvent accessibility of the wild-type side chain.

Amino Acid Sequence↗
PubMed1988

The Mnt repressor of bacteriophage P22: role of C-terminal residues in operator binding and tetramer formation.

A set of C-terminal deletion mutants of the Mnt repressor of bacteriophage P22 has been constructed, and the corresponding truncated proteins have been purified. A truncated protein lacking the three C-terminal residues, Lys80-Thr81-Thr82, binds operator DNA with an affinity near wild type and has a normal tetrameric structure. Loss of the next residue, Lys79, causes a 600-fold decrease in operator affinity, but the truncated protein is still tetrameric. Further sequential deletions of Tyr78 and Leu77 cause modest decreases in operator affinity, but the truncated proteins are now dimeric. These results indicate that Lys79 is an important determinant of the high affinity of Mnt repressor for operator DNA and that Tyr78 is an important determinant of tetramer formation by Mnt repressor.

Amino Acid Sequence↗
PubMed1988

Flexibility of the yeast alpha 2 repressor enables it to occupy the ends of its operator, leaving the center free.

The yeast alpha 2 protein, the product of the MAT alpha 2 gene, is a regulator of yeast cell type; it turns off transcription of the a-specific genes by binding to an operator located upstream of each gene. In this paper we describe the domain structure, subunit organization, and some unusual features of the way this protein contacts its operator. We show that the protein is folded into two domains. The carboxy-terminal domain binds specifically to the operator; the amino-terminal domain contains dimerization contacts. The alpha 2 dimer differs from those of the phage repressors in that it is flexible and therefore is able to bind tightly to differently spaced operator half-sites. In the natural operator, the centers of the operator half-sites are two and one-half turns of DNA apart, exposing them on opposite sides of the DNA helix. We show that the design of alpha 2 allows a dimer to reach across its operator such that it occupies the two half-sites but leaves the middle of the operator available to other proteins.

Base Sequence↗
PubMed1987

Bacteriophage P22 Mnt repressor. DNA binding and effects on transcription in vitro.

We have examined the binding of Mnt repressor to operator DNA in vitro and have determined how this binding affects the level of transcription from two nearby promoters, Pant and Pmnt. Mnt binds to a region of DNA that overlaps the startpoint of transcription of Pant and the -35 region of Pmnt. Mnt represses transcription in vitro from Pant and enhances transcription from Pmnt. Protection and interference experiments show that Mnt binds to a single, 17 base-pair operator site. The operator sequence and the protein-DNA contacts are symmetric. Mnt makes major groove contacts on both faces of the operator DNA. At pH 7.5, 200 mM-KCl, 22 degrees C, the Mnt tetramer binds operator with high affinity (Kd = 2.2 X 10(-11M) and the protein-DNA complex is quite stable (t1/2 = 48 min). Operator binding shows large dependencies on pH, salt concentration, and temperature.

Base Sequence↗
PubMed1987

Interaction of the bacteriophage P22 Arc repressor with operator DNA.

Are repressor binds to a single, partially symmetric, 21 base-pair operator site that is centered between the -10 and -35 regions of the Pant promoter. Protection and interference experiments show that Arc makes contacts with the operator on one side of the DNA helix. Although Arc is a small protein (53 residues/subunit), it makes contacts that are farther from the center of the operator than those made by many larger repressors. These extended contacts include the phosphate groups at the ends of the 21 base-pair site. Under standard conditions (pH 7.5, 100 mM-KCl, 3 mM-MgCl2, 22 degrees C) half-maximal operator binding is observed at an Arc concentration of 2.5 X 10(-9) M and the protein-DNA complex is very stable (t1/2 approximately equal to 80 min).

DNA, Viral↗