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R T Sauer

Publications and source records attributed to R T Sauer.

At least 19 recordsLinked to original sources
PubMed1992

Structural and energetic consequences of disruptive mutations in a protein core.

We have characterized the properties of a set of variants of the N-terminal domain of lambda repressor bearing disruptive mutations in the hydrophobic core. These mutations include some that dramatically alter the total core residue volume (by up to six methylene groups) and some that place a single polar residue into the otherwise hydrophobic core. The structural properties of the purified proteins have been studied by CD spectroscopy, biological activity, recognition by conformation-specific monoclonal antibodies, and 1H NMR spectroscopy. The stabilities of the proteins have been measured by thermal and guanidine hydrochloride denaturation. Proteins with disruptive core mutations are found to display a continuum of increasingly nonnative properties. Large internal volume changes cause both significant conformational rearrangements and destabilization by up to 5 kcal/mol. Variants with polar substitutions at core positions no longer behave like well-folded proteins but rather display characteristics of molten globules. However, even proteins bearing some of the most disruptive mutations retain many of the crude secondary and tertiary structural features of the wild-type protein. These results indicate that primitive elements of native structure can form in the absence of normal core packing.

Amino Acid Sequence↗
PubMed1992

Tsp: a tail-specific protease that selectively degrades proteins with nonpolar C termini.

An Escherichia coli protease designated Tsp (tail-specific protease) has been purified, and its gene has been cloned and sequenced. Tsp specifically degrades a variant of the N-terminal domain of lambda repressor in which the five C-terminal residues, which are polar in wild type, have been replaced by nonpolar residues. This substrate specificity in vitro parallels the previously reported selective degradation in vivo of N-terminal-domain variants with nonpolar C-terminal residues. The gene sequence and N-terminal protein sequence of Tsp predict a protein of 660 amino acids. The deduced protein sequence of Tsp shows no significant homology to known protease sequences but does show sequence similarity to the human and bovine interphotoreceptor retinoid-binding proteins, which bind hydrophobic ligands.

Amino Acid Sequence↗
PubMed1992

Biochemical and genetic analysis of operator contacts made by residues within the beta-sheet DNA binding motif of Mnt repressor.

Residues 2, 6, 8 and 10 of Mnt repressor are the major determinants of operator DNA binding and recognition. Here, we investigate the interaction of wild-type Mnt and mutants bearing the Arg2----Lys, His6----Ala, Asn8----Ala and Arg10----Lys mutations with operator DNA modified by methylation or by symmetric base substitutions. The wild-type pattern of methylation interference is altered in specific ways for each of the mutant proteins. In addition, some of the mutant proteins show a 'loss of contact' phenotype with specific mutant operators. Taken together, these and previous results predict the following contacts between side chains in the Mnt tetramer and operator DNA: Arg2 recognizes the guanines at operator positions 10 and 12; His6 contacts the guanines at operator positions 5 and 17; Asn8 contacts operator positions 4, 7, 15 and 18; Arg10 contacts the guanines at operator positions 8 and 14. The proposed contacts can be accommodated in a structural model in which the anti-parallel beta-sheet motifs of Mnt dimers lie in the major grooves of each operator half-site, centered over pseudo-symmetry axes that are 5.5 bp from the central dyad axis of the operator.

Base Sequence↗
PubMed1991

The role of internal packing interactions in determining the structure and stability of a protein.

Cassette mutagenesis has been used to investigate how internal packing interactions help to specify a protein's three-dimensional structure and stability. Three interacting residues in the hydrophobic core of the N-terminal domain of lambda repressor were randomized combinatorially. The randomization was restricted to the five amino acids Val, Leu, Ile, Met and Phe, thereby generating a sterically diverse set of core sequences composed solely of hydrophobic residues. We have isolated 78 of the 125 possible sequences generated by this randomization. Approximately 70% of the isolated sequences show some level of biological activity, and thus still carry sufficient information to encode the basic structure of lambda repressor. An assay based on the temperature dependence of activity in vivo has been used to estimate the relative activities and thermal stabilities of the set of mutants. In addition, nine mutants have been purified and their stabilities and DNA binding activities characterized in vitro. Of the 56 active sequences, only two, in addition to the wild-type, maintain the wild-type level of stability and activity. All three of these proteins satisfy stringent requirements for specifically shaped residues at each position. All of the remaining active sequences have reduced stabilities and/or reduced DNA binding affinities. These and previous results suggest that there are two levels of structural information encoded in core residues. At the first level, the basic structural information appears to reside largely in the hydrophobic character of these residues. The majority of sequences that simply maintain hydrophobicity at core positions are able to adopt the overall lambda repressor fold and maintain moderate stability. At the second, more detailed level, specific steric features of these residues and their packing interactions clearly act as important determinants of the protein's precise structure and stability. These results imply that many of the basic structural features of a protein could be predicted from relatively simple, degenerate sequence patterns.

DNA, Bacterial↗
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↗