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R T Sauer

Publications and source records attributed to R T Sauer.

At least 73 records · Page 4Linked to original sources
PubMed1996

Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease.

The degQ and degS genes of Escherichia coli encode proteins of 455 and 355 residues, respectively, which are homologs of the DegP protease. The purified DegQ protein has the properties of a serine endoprotease and is processed by the removal of a 27-residue amino-terminal signal sequence. A plasmid expressing degQ rescues the temperature-sensitive phenotype of a strain bearing the degP41 deletion, implying that DegQ, like DegP, functions as a periplasmic protease in vivo. Deletions in the degQ gene cause no obvious growth defect, while those in the degS gene result in a small-colony phenotype. The latter phenotype is rescued by a plasmid expressing the degS gene but not by plasmids expressing the degQ or degP genes. This result and the inability of a plasmid expressing degS to rescue the temperature-sensitive degP41 phenotype indicate that the DegS protein is functionally different from the DegQ and DegP proteins.

Amino Acid Sequence↗
PubMed1995

Identification of active site residues of the Tsp protease.

In a search for active-site residues of the Tsp protease, 20 positions were individually mutated to alanine, the mutant strains were assayed for growth defects in vivo, and the purified proteins were assayed for proteolytic activity in vitro. Alanine substitutions at three positions, Ser-430, Asp-441, and Lys-455, result in inactive proteases that have structures and substrate-binding properties similar to wild type, suggesting that the side chains at these positions participate in catalysis. Replacing Ser-430 with cysteine results in a partially active protease, which is inhibited by cysteine-modifying reagents. Replacing Asp-441 with asparagine does not significantly affect activity. However, other residues, including histidine and arginine, cannot functionally replace Lys-455. These data are consistent with a serine-lysine dyad mechanism, similar to those proposed for the LexA-like proteases, the type I signal peptidases, and the class A beta-lactamases.

Amino Acid Sequence↗
PubMed1995

Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex.

To assess the importance of minor-groove and major-groove interactions in homeodomain-DNA recognition, the binding properties of variants of the altered-specificity engrailed homeodomain, containing Lys50, and its DNA site TAATCC were determined. This homeodomain contacts bases in the minor groove of the DNA using Arg3 and Arg5 from its N-terminal arm and contacts bases in the major groove of the DNA using Ile47, Lys50, and Asn51 from its third alpha-helix. Mutation of Arg3 or Ile47 to alanine reduces binding affinity 10-20-fold while mutation of Arg5, Asn51, or Lys50 to alanine reduces binding affinity > 100-fold, indicating that both minor-groove and major-groove interactions contribute to the overall binding energy. Binding site selections and affinity measurements show that the homeodomain can also discriminate among different base pairs in the minor groove and the major groove. However, the interactions between Lys50 of the recognition helix and the major-groove edges of base pairs 5 and 6 are more specific than interactions mediated by Arg3 and Arg5 in the N-terminal arm and the minor-groove edges of base pairs 1 and 2.

Alanine↗
PubMed1995

P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and refolding.

The kinetics of unfolding and refolding have been measured for a set of Arc repressor mutants bearing single amino acid substitutions at 44 of the 53 residue positions. Roughly half of the mutations cause significant changes in the unfolding and/or refolding rate constants. These substitutions alter the hydrophobic core, tertiary hydrogen bonds and salt bridges, and glycines with restricted backbone conformations. Overall, the mutations cause larger changes in the unfolding rates than the refolding rates, indicating that significantly less side-chain information is used between the denatured state and transition state than between the transition state and native state. The set of mutants displays reasonable Brønsted behavior, suggesting that many native interactions are partially formed in the transition state. Taken together, these observations suggest that the overall structure of most of the protein must be somewhat native-like in the transition state but without close, complementary packing of the hydrophobic core or good hydrogen bond geometry. Such a transition state is inconsistent with a model in which monomers fold to their correct conformations and then dock to form the dimer but supports a model in which folding and dimerization are concurrent processes.

Bacteriophage P22↗
PubMed1995

Domains of Mnt repressor: roles in tetramer formation, protein stability, and operator DNA binding.

The Mnt repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of gene regulatory proteins. Proteolytic cleavage of Mnt with chymotrypsin reveals that it consists of two structural domains. Both domains are required for high-affinity operator binding. The N domain (residues 1-51) is dimeric and binds weakly but specifically to operator DNA. The C domain (residues 52-82) forms an independent alpha-helical, tetramerization domain and, by itself, has no DNA-binding activity. In intact Mnt, the N and C domains help to stabilize each other against denaturation but appear to be linked rather flexibly. Assays of the half-operator affinities of Mnt and the isolated N domain indicate that binding to adjacent half-sites in the whole operator is stabilized by protein-protein contacts between N domains in addition to protein-protein contacts between C domains.

Allosteric Regulation↗
PubMed1995

P22 Arc repressor: role of cooperativity in repression and binding to operators with altered half-site spacing.

Dimers of P22 Arc repressor bind to half-sites of the 21 bp arc operator and interact cooperatively to stabilize a DNA-bound tetramer. Mutation of Ser35 (a residue in the dimer-dimer interface) to Arg or Leu disrupts cooperative binding. The mutant proteins have near wild-type stabilities, give operator footprints like wild-type, and prevent binding of RNA polymerase to the Pant promoter in vitro. These mutants are, however, largely inactive in vivo. Thus, although cooperativity is not structurally required for repression, it appears that the additional DNA-binding energy from dimer-dimer cooperativity is required for normal biological function. Altering the spacing between the DNA half-sites by even one base-pair eliminates dimer-dimer cooperativity, indicating that Arc dimers need to be oriented correctly by half-site binding to allow the interactions that stabilize the tetrameric complex.

Bacteriophage P22↗
PubMed1995

Critical side-chain interactions at a subunit interface in the Arc repressor dimer.

In the Arc repressor dimer, the side chains of Ile37 and Val41 in alpha-helix B pack against each other and against the symmetry-related side chains of Ile37' and Val41' in alpha-helix B' to form part of the hydrophobic core and the dimer interface. Following combinatorial mutagenesis of these positions, only the wild-type combination of hydrophobic residues was recovered as a fully active protein, and only a few conservative replacements were recovered as stably folded or partially active proteins. Equilibrium and kinetic studies of the folding of purified mutants show that the delta-CH3 groups of Ile37 and Ile37' contribute approximately 2 kcal/mol of dimer to protein stability and are involved in interactions that are only partially formed in the transition state for protein folding. Alanine substitution at either position 37 or 41 results in proteins which differ from wild type in being monomeric at a concentration of 10 microM, having reduced secondary structure, having solvent-exposed tryptophans, and showing non-cooperative thermal and urea denaturation transitions. These mutants appear to exist in a physiologically denatured state that is similar in many ways to the molten globule state.

Alanine↗
PubMed1995

Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8.

Arc repressor is a small, dimeric DNA-binding protein that belongs to the ribbon-helix-helix family of transcription factors. Replacing Pro8 at the N-terminal end of the beta-sheet with leucine increases the stability of the mutant protein by 2.5 kcal/mol of dimer. However, this enhanced stability is achieved at the expense of significantly reduced DNA binding affinity. The structure of the PL8 mutant dimer has been determined to 2.4-A resolution by X-ray crystallography. The overall structure of the mutant is very similar to wild type, but Leu8 makes an additional interstrand hydrogen bond at each end of the beta-sheet of the mutant, increasing the total number of beta-sheet hydrogen bonds from six to eight. Comparison of the refolding and unfolding kinetics of the PL8 mutant and wild-type Arc shows that the enhanced stability of the mutant is accounted for by a decrease in the rate of protein unfolding, suggesting that the mutation acts to stabilize the native state and that the beta-sheet forms after the rate-limiting step in folding. The reduced operator affinity of the PL8 dimer appears to arise because the mutant cannot make the new interstrand hydrogen bonds and simultaneously make the wild-type set of contacts with operator DNA.

Crystallography, X-Ray↗
PubMed1995

C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease.

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.

Amino Acid Sequence↗
PubMed1995

Are buried salt bridges important for protein stability and conformational specificity?

The side chains of Arg 31, Glu 36 and Arg 40 in Arc repressor form a buried salt-bridge triad. The entire salt-bridge network can be replaced by hydrophobic residues in combinatorial randomization experiments resulting in active mutants that are significantly more stable than wild type. The crystal structure of one mutant reveals that the mutant side chains pack against each other in an otherwise wild-type fold. Thus, simple hydrophobic interactions provide more stabilizing energy than the buried salt bridge and confer comparable conformational specificity.

Alanine↗
PubMed1995

Cooperatively folded proteins in random sequence libraries.

The structural properties of proteins recovered from random sequence libraries can be used to investigate the relationship between folding and sequence information. Here, we show that helical proteins displaying cooperative thermal denaturation transitions can be easily recovered from a library containing 80-residue proteins predominantly composed of glutamine, leucine, and arginine, with an average hydrophobicity level similar to that of natural proteins. The native structure of one of these proteins has a stability and oligomeric form similar to that of many natural proteins but differs in having no slowly exchanging amide hydrogens.

Amino Acid Sequence↗
PubMed1995

Dramatic changes in DNA-binding specificity caused by single residue substitutions in an Arc/Mnt hybrid repressor.

Arc and Mnt are homologous repressors which recognize operator sequences that differ at 8-10 important positions. Nevertheless, single residue changes in an Arc/Mnt hybrid protein can switch DNA-binding specificity between the two operators and even allow one particular hybrid to bind strongly to both operators. The ability of single residue changes to radically alter binding specificity involves: 'master' residues that mediate some base contacts directly and some base contacts indirectly through residue-residue hydrogen bonds; identical residues which can make alternative sets of DNA contacts in the two operators; and amplification of the effect of each mutation because the proteins bind operator DNA as tetramers.

Amino Acid Sequence↗
PubMed1994

Solution structure of dimeric Mnt repressor (1-76).

Wild-type Mnt repressor of Salmonella bacteriophage P22 is a tetrameric protein of 82 residues per monomer. A C-terminal deletion mutant of the repressor denoted Mnt (1-76) is a dimer in solution. The structure of this dimer has been determined using NMR. The NMR assignments of the majority of the 1H, 15N, and 13C resonances were obtained using 2D and triple-resonance 3D techniques. Elements of secondary structure were identified on the basis of characteristic sequential and medium range NOEs. For the structure determination more than 1000 NOEs per monomer were obtained, and structures were generated using distance geometry and restrained simulated annealing calculations. The discrimination of intra- vs intermonomer NOEs was based upon the observation of intersubunit NOEs in [15N,13C] double half-filtered NOESY experiments. The N-terminal part of Mnt (residues 1-44), which shows a 40% sequence homology with the Arc repressor, has a similar secondary and tertiary structure. Mnt (1-76) continues with a loop region of irregular structure, a third alpha-helix, and a random coil C-terminal peptide. Analysis of the secondary structure NOEs, the exchange rates, and the backbone chemical shifts suggests that the carboxy-terminal third helix is less stable than the remainder of the protein, but the observation of intersubunit NOEs for this part of the protein enables the positioning of this helix. The rsmd's between the backbone atoms of the N-terminal part of the Mnt repressor (residues 5-43, 5'-43') and the Arc repressor is 1.58 A, and between this region and the corresponding part of the MetJ repressor 1.43 A.

Amino Acid Sequence↗
PubMed1994

Differential DNA-binding specificity of the engrailed homeodomain: the role of residue 50.

To assess the importance of residue 50 in determining the binding specificity of the homeodomain from the engrailed transcription factor of Drosophila, the DNA-binding properties of isolated homeodomains containing glutamine (wild type), alanine, and lysine at this position have been studied. In binding site selection experiments using the wild-type engrailed homeodomain, TAATTA was identified as a high-affinity, consensus binding site. When the glutamine at position 50 was replaced by a lysine (QK50), the binding site preference changed to TAATCC. The half-life and affinity of the complex between the QK50 protein and a DNA site containing TAATCC were increased significantly compared to the half-life and affinity of the complex between the wild-type protein and a TAATTA site. This suggests that Lys50 forms a more favorable interaction with the TAATCC DNA than Gln50 does with the TAATTA site. In fact, the wild-type Gln50 side chain (which forms a hydrophobic interaction with the last A:T base pair of the TAATTA site in the cocrystal structure [Kissinger, C. R., Liu, B., Martin-Blanco, E., Kornberg, T. B., & Pabo, C. O. (1990) Cell 63, 579-590]) appears to play only a small role in determining binding affinity and specificity for the TAATTA site, as the QA50 mutant has only a 2-fold reduced affinity for the TAATTA site and discriminates between the TAATTA and TAATCC sites as well as the wild-type protein. As a result, determinants in addition to Gln50 must be involved in establishing the differential binding specificity of the engrailed homeodomain.

Amino Acid Sequence↗
PubMed1994

A novel class of FokI restriction endonuclease mutants that cleave hemi-methylated substrates.

A genetic screen was used to identify amino acid substitutions that enable the FokI restriction endonuclease to cleave DNA in cells that express the cognate methyltransferase activity. Missense mutations that give rise to this phenotype were isolated at eight different positions (G188K, P196S, T343I, S388N, S395F, E407K, E410K, D421N), clustered in two regions of the polypeptide sequence of FokI. Two of the mutant endonucleases (P196S and D421N) were purified to homogeneity and analyzed in detail. Both mutants cleave FokI target sites (5'-GGATG-3') in a manner similar to the wild-type enzyme. Neither mutant cleaved noncanonical sequences, but both efficiently cleaved DNA substrates containing hemi-methylated FokI sites. This class of mutations has not been observed with other restriction enzymes.

Base Sequence↗
PubMed1994

Folded proteins occur frequently in libraries of random amino acid sequences.

A library of synthetic genes encoding 80- to 100-residue proteins composed mainly of random combinations of glutamine (Q), leucine (L), and arginine (R) has been expressed in Escherichia coli. These genes also encode an epitope tag and six carboxyl-terminal histidines. Screening of this library by immunoblotting showed that 5% of these QLR proteins are expressed at readily detectable levels. Three well-expressed QLR proteins were purified and characterized. Each of these proteins has significant alpha-helical content, is largely resistant to degradation by Pronase, and has a distinct oligomeric structure. In addition, one protein unfolds in a highly cooperative manner. These properties of the QLR proteins demonstrate that they possess folded structures with some native-like properties. The QLR proteins differ from most natural proteins, however, in being remarkably resistant to denaturant-induced and thermal-induced unfolding and in being relatively insoluble in the absence of denaturants.

Amino Acid Sequence↗
PubMed1994

DNA recognition by beta-sheets in the Arc repressor-operator crystal structure.

Transcription of the ant gene during lytic growth of bacteriophage P22 (ref. 1) is regulated by the cooperative binding of two Arc repressor dimers to a 21-base-pair operator site. Here we report the co-crystal structure of this Arc tetramer-operator complex at 2.6 A resolution. As expected from genetic and structural studies and from the co-crystal structure of the homologous Escherichia coli MetJ repressor, each Arc dimer uses an antiparallel beta-sheet to recognize bases in the major groove. However, the Arc and MetJ complexes differ in several important ways: the beta-sheet-DNA interactions of Arc are far less symmetrical; DNA binding by Arc is accompanied by important conformational changes in the beta-sheet; and Arc uses a different part of its protein surface for dimer-dimer interactions.

Base Sequence↗