PubMed Health⌕ Search

Biomedical subjects

R T Sauer

Publications and source records attributed to R T Sauer.

At least 109 records · Page 6Linked to original sources
PubMed1990

Arc repressor is tetrameric when bound to operator DNA.

The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions.

A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.

Amino Acid Sequence↗
PubMed1990

An essential proline in lambda repressor is required for resistance to intracellular proteolysis.

Pro78 is a solvent-exposed residue at the N-terminal end of alpha-helix 5 in the DNA binding domain of lambda repressor. Random mutagenesis experiments have suggested that Pro78 is essential [Reidhaar-Olson, J.F., & Sauer, R.T. (1990) Proteins: Struct., Funct., Genet. (in press)]. To investigate the requirement for proline at this position, we constructed and studied the properties of a set of ten position 78 mutant proteins. All of these mutants have decreased intracellular activities and are expressed at significantly lower levels than wild type. Pulse-chase experiments show that the mutant proteins are rapidly degraded in the cell; the mutants examined had half-lives of 11-35 min, whereas the wild-type protein has a half-life of greater than 10 h. The rapid degradation of position 78 mutants is not suppressed by mutations that affect known Escherichia coli proteases. The Pro78----Ala mutant could be overexpressed in a dnaJ- strain and was purified. This mutant has full DNA binding activity in vitro, suggesting that its folded structure and ability to form active dimers are similar to those of wild type. The PA78 mutant (Tm = 48 degrees C) is less thermally stable than wild type (Tm = 55 degrees C). Double-mutant studies show that this instability contributes to but is not the main cause of its rapid intracellular degradation and also suggest that proteolysis proceeds from the denatured forms of proteins containing the PA78 substitution. The PA78 mutation does not appear to introduce a new cleavage site for cellular proteases, nor does the mutation enhance susceptibility to proteases such as thermolysin and trypsin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface.

It is rare for amino-acid substitutions on the surface of proteins to have large stabilizing or destabilizing effects. Nevertheless, one substitution of this type, the Tyr 26----Cys mutation in lambda Cro, increases the melting temperature of the protein by 11 degrees C and the stability by 2.2 kcal mol-1. Here we show that the stability of Cro can be increased by many different amino-acid substitutions at position 26, with increasing stability showing a good correlation with decreasing side-chain hydrophobicity. As Tyr 26 is hyper-exposed to solvent in the Cro crystal structure, we suggest that wild-type and variant proteins with other hydrophobic side chains at position 26 are destabilized as a result of a reverse hydrophobic effect caused by the side chain being more exposed to solvent in the native than in the denatured state.

DNA Mutational Analysis↗
PubMed1990

Deciphering the message in protein sequences: tolerance to amino acid substitutions.

An amino acid sequence encodes a message that determines the shape and function of a protein. This message is highly degenerate in that many different sequences can code for proteins with essentially the same structure and activity. Comparison of different sequences with similar messages can reveal key features of the code and improve understanding of how a protein folds and how it performs its function.

Amino Acid Sequence↗
PubMed1990

Identification of protein folds: matching hydrophobicity patterns of sequence sets with solvent accessibility patterns of known structures.

Hydrophobic side chains often are buried in the interior of a protein, and evolutionarily related proteins usually maintain the hydrophobic character of buried positions. In this paper we show that a pattern of hydrophobicity values derived from a set of related protein sequences is well correlated with the linear pattern of side-chain solvent accessibility values, calculated from a known protein structure representative of the sequences. In several cases, information from aligned sequences can be used to select the correct tertiary fold from a large data base of protein structures.

Algorithms↗
PubMed1990

Functionally acceptable substitutions in two alpha-helical regions of lambda repressor.

A method of targeted random mutagenesis has been used to investigate the informational content of 25 residue positions in two alpha-helical regions of the N-terminal domain of lambda repressor. Examination of the functionally allowed sequences indicates that there is a wide range in tolerance to amino acid substitution at these positions. At positions that are buried in the structure, there are severe limitations on the number and type of residues allowed. At most surface positions, many different residues and residue types are tolerated. However, at several surface positions there is a strong preference for hydrophilic amino acids, and at one surface position proline is absolutely conserved. The results reveal the high level of degeneracy in the information that specifies a particular protein fold.

Amino Acid Sequence↗
PubMed1990

Carboxy-terminal determinants of intracellular protein degradation.

Using the amino-terminal domain of lambda repressor as a model system, we show that residues in an unstructured region at the extreme carboxyl terminus of the protein are important for determining its proteolytic susceptibility in Escherichia coli. Nonpolar amino acids are destabilizing when placed at the 5 carboxy-terminal residue positions, whereas charged and polar residues are stabilizing. The stabilizing effect of a single charged residue is greatest when it is at the terminal position and diminishes with increasing distance from the carboxyl terminus. The position of destabilizing sequences with respect to the free carboxyl terminus is important for their effect, but their distance from the folded portion of the protein is not important. Specific degradation of proteins with nonpolar carboxyl termini has been reconstituted in vitro using a partially pure, soluble fraction. This degradation is not ATP-dependent. Moreover, amino-terminal domain variants with nonpolar carboxy-terminal residues are still rapidly degraded in strains that are deficient in proteolysis of abnormal proteins. These data suggest that the degradation of amino-terminal domain variants with nonpolar carboxy-terminal residues involves proteolytic components distinct from those known to be important for the turnover of unfolded proteins in E. coli.

Amino Acid Sequence↗
PubMed1989

NMR studies of Arc repressor mutants: proton assignments, secondary structure, and long-range contacts for the thermostable proline-8----leucine variant of Arc.

Arc repressor is a 53-residue sequence-specific DNA binding protein. We report the assignment of the proton NMR spectrum and the secondary structure for the thermostable PL8 variant of Arc. This mutant, which differs from wild type by a Pro-8----Leu substitution, was chosen for study because its enhanced stability allows spectra to be acquired at elevated temperatures where spectral resolution is higher. The first five residues of the protein play important roles in DNA binding but appear to be disordered in solution. Residues 6-14 form the remaining part of the N-terminal DNA binding region of the protein and assume an antiparallel beta-conformation. This indicates that Arc is a member of a new class of DNA binding proteins. The observed interresidue nuclear Overhauser effects are consistent with a beta-strand, gamma-turn, beta-strand structure for the residue 6-14 region, although other structures are also consistent with the data. The remaining portion of the protein is predominantly alpha-helical. Residues 16-26 and 35-50 form amphipathic alpha-helices which may pack together in a four-helix bundle in the protein dimer.

Amino Acid Sequence↗
PubMed1989

Equilibrium dissociation and unfolding of the Arc repressor dimer.

The equilibrium unfolding reaction of Arc repressor, a dimeric DNA binding protein encoded by bacteriophage P22, can be monitored by fluorescence or circular dichroism changes. The stability of Arc is concentration dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The stability of the protein is decreased at low pH and increased by high salt concentration. The salt dependence suggests that two ions bind preferentially to the folded protein. In 10 mM potassium phosphate (pH 7.3) and 100 mM KCl, the unfolding free energy reaches a maximum near room temperature. The results suggest that at the low protein concentrations where operator DNA binding is normally measured, Arc is predominantly monomeric and unfolded.

Circular Dichroism↗
PubMed1989

Identification of functionally important residues in the DNA binding region of the mnt repressor.

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.

Amino Acid Sequence↗