Biomedical subjects

R T Sauer

Publications and source records attributed to R T Sauer.

At least 37 records · Page 2Linked to original sources

PubMed2000

An evolutionary bridge to a new protein fold.

Arc repressor bearing the N11L substitution (Arc-N11L) is an evolutionary intermediate between the wild type protein, in which the region surrounding position 11 forms a beta-sheet, and a double mutant 'switch Arc', in which this region is helical. Here, Arc-N11L is shown to be able to adopt either the wild type or mutant conformations. Exchange between these structures occurs on the millisecond time scale in a dynamic equilibrium in which the relative populations of each fold depend on temperature, solvent conditions and ligand binding. The N11L mutation serves as an evolutionary bridge from the beta-sheet to the helical fold because in the mutant, Leu is an integral part of the hydrophobic core of the new structure but can also occupy a surface position in the wild type structure. Conversely, the polar Asn 11 side chain serves as a negative design element in wild type Arc because it cannot be incorporated into the core of the mutant fold.

Amino Acid Sequence↗

PubMed1999

SsrA-mediated peptide tagging caused by rare codons and tRNA scarcity.

SsrA RNA mediates the addition of a C-terminal peptide tag (AANDENYALAA) to bacterial proteins translated from mRNAs without in-frame stop codons. This process involves both tRNA- and mRNA-like functions of SsrA and targets the tagged proteins for degradation. By designing an SsrA variant that adds a peptide tag (AANDENYALDD) that does not result in rapid degradation, we show that tagging of a model protein synthesized from an mRNA without stop codons can be detected both in vivo and in vitro. We also use this assay to demonstrate that ribosome stalling at clusters of rare arginine codons in mRNA is sufficient to recruit and activate the SsrA peptide tagging system. An essential requirement for tagging at rare AGA codons is a scarcity of the cognate tRNA; supplemental tRNA(AGA) suppresses tagging, and depleting the available pool of tRNA(AGA) enhances tagging and reveals tagging caused by single rare AGA codons. Protein tagging at sites corresponding to rare codons appears to involve SsrA action at an internal mRNA site rather than at the 3' end of a cleaved mRNA.

Amino Acid Sequence↗

PubMed1999

SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA).

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of the partially synthesized nascent polypeptide chain. The SsrA-tagged protein is then degraded by C-terminal-specific proteases. SmpB, a unique RNA-binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality-control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA-defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB-SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild-type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

Alanine↗

PubMed1999

The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex.

The toxin (Doc) and antidote (Phd) proteins of the plasmid addiction system of bacteriophage P1 were purified as a complex. Cocrystals of the complex contained a 2:1 molar ratio of Phd:Doc as assayed by dye binding following SDS-polyacrylamide gel electrophoresis and as determined by amino acid analysis. Gel filtration and analytical ultracentrifugation revealed that the two addiction proteins interact in solution to form a P2D trimer composed of one Doc and two Phd molecules. These results support a model in which Phd inhibits the toxic activity of Doc by direct binding. Circular dichroism experiments showed that changes in secondary structure accompany formation of the heterotrimeric complex, raising the possibility that Phd may act by an allosteric mechanism. Studies of Phd and Doc molecules labeled with fluorescent energy donor and acceptor groups gave an equilibrium dissociation constant of about 0.8 microM(2) and a very short, sub second half-life of complex dissociation. As a consequence, low concentrations of free Doc toxin are likely to be present both transiently and in the steady state in cells containing the Phd antidote, making mechanisms of single-hit Doc toxicity improbable.

Bacteriophage P1↗

PubMed1999

Lon and Clp family proteases and chaperones share homologous substrate-recognition domains.

Lon protease and members of the Clp family of molecular chaperones and protease regulatory subunits contain homologous regions with properties expected for substrate-binding domains. Fragments corresponding to these sequences are stably and independently folded for Lon, ClpA, and ClpY. The corresponding regions from ClpB and ClpX are unstable. All five fragments exhibit distinct patterns of binding to three proteins that are protease substrates in vivo: the heat shock transcription factor sigma32, the SOS mutagenesis protein UmuD, and Arc repressor bearing the SsrA degradation tag. Recognition of UmuD is mediated through peptide sequences within a 24-residue N-terminal region whereas recognition of both sigma32 and SsrA-tagged Arc requires sequences at the C terminus. These results indicate that the Lon and Clp proteases use the same mechanism of substrate discrimination and suggest that these related ATP-dependent bacterial proteases scrutinize accessible or disordered regions of potential substrates for the presence of specific targeting sequences.

ATP-Dependent Proteases↗

PubMed1999

The solution structure and dynamics of an Arc repressor mutant reveal premelting conformational changes related to DNA binding.

The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.

DNA↗

PubMed1999

Evolution of a protein fold in vitro.

A "switch" mutant of the Arc repressor homodimer was constructed by interchanging the sequence positions of a hydrophobic core residue, leucine 12, and an adjacent surface polar residue, asparagine 11, in each strand of an intersubunit beta sheet. The mutant protein adopts a fold in which each beta strand is replaced by a right-handed helix and side chains in this region undergo significant repacking. The observed structural changes allow the protein to maintain solvent exposure of polar side chains and optimal burial of hydrophobic side chains. These results suggest that new protein folds can evolve from existing folds without drastic or large-scale mutagenesis.

Amino Acid Sequence↗

PubMed1999

Tolerance of Arc repressor to multiple-alanine substitutions.

Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure.

Alanine↗

PubMed1999

Origins of DNA-binding specificity: role of protein contacts with the DNA backbone.

A central question in protein-DNA recognition is the origin of the specificity that permits binding to the correct site in the presence of excess, nonspecific DNA. In the P22 Arc repressor, the Phe-10 side chain is part of the hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor-operator complex. Characterization of a library of position 10 variants reveals that Phe is the only residue that results in fully active Arc. One class of mutants folds stably but binds operator with reduced affinity; another class is unstable. FV10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well. The affinity differences between FV10 and wild type indicate that each Phe-10 side chain contributes 1.5-2.0 kcal to operator binding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity. This appears to be a direct contribution as the crystal structure of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex.

Amino Acid Sequence↗

PubMed1999

Stability and DNA binding of the phd protein of the phage P1 plasmid addiction system.

The plasmid addiction module of bacteriophage P1 encodes two proteins, Doc, a toxin that is stable to proteolytic degradation, and Phd, the toxin's antidote that is proteolytically unstable. Phd has been shown to autoregulate its expression by specific DNA binding. Here, we investigate the secondary structure and thermal stability of Phd, the effect of operator DNA binding on the structure and stability of Phd, and the stoichiometry, affinity, and cooperativity of Phd binding to operator subsites and intact operator DNA. Phd folds as a monomer at low temperatures or in the presence of osmolytes but exists predominantly in an unfolded conformation at 37 degreesC. The native state of Phd is stabilized by operator binding. Two Phd monomers bind to each operator subsite, and four monomers bind to the intact operator. The subsite binding reaction shows a second-order dependence on protein concentration and monomer-bound DNA species are unpopulated, suggesting that two Phd molecules bind cooperatively to each operator subsite. In intact operator binding experiments, both dimer-bound and tetramer-bound DNA species are populated, and binding occurs at protein concentrations similar to those required for subsite binding, suggesting that there is no significant dimer-dimer cooperativity.

Bacteriophage P1↗

PubMed1999

The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.

Amino Acid Sequence↗

PubMed1999

Acceleration of the refolding of Arc repressor by nucleic acids and other polyanions.

The refolding rate of the Arc repressor dimer can be accelerated 30-fold or more by negatively charged polymers including single-stranded and double-stranded DNA, RNA, and polyvinylsulfate but not by neutral or positively charged polymers. The salt-dependence of the polyanion-mediated process and mutant studies indicate that electrostatic interactions are important in the rate acceleration. Urea-dependence studies suggest that Arc is relatively unstructured in the transition state for polyanion-stimulated refolding. At low ionic strength, the observed kinetics of refolding are consistent with a model in which denatured Arc monomers bind rapidly and nonspecifically to the polyanion and complete folding in the bound state.

Animals↗

PubMed1999

NikR is a ribbon-helix-helix DNA-binding protein.

Escherichia coli NikR, a repressor with homologs in other bacteria and archaea, was identified as a potential new member of the ribbon-helix-helix (beta-alpha-alpha) family of transcription factors in profile based sequence searches and in structure prediction experiments. Biophysical and biochemical characterization of the N-terminal domain of NikR show that it has many features expected of a beta-alpha-alpha protein including alpha-helical content, dimeric solution form, concentration dependent thermal stability, and ability to bind DNA in sequence-specific manner. Mutation of a residue predicted to be important for DNA-binding reduces operator affinity but does not affect the secondary structure or stability of the protein.

Amino Acid Sequence↗

PubMed1999

Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions.

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.

Amino Acids↗

PubMed1998

Optimizing the stability of single-chain proteins by linker length and composition mutagenesis.

Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 microM-10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined. In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.

Gene Library↗

PubMed1998

The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system.

Interruption of translation in Escherichia coli can lead to the addition of an 11-residue carboxy-terminal peptide tail to the nascent chain. This modification is mediated by SsrA RNA (also called 10Sa RNA and tmRNA) and marks the tagged polypeptide for proteolysis. Degradation in vivo of lambda repressor amino-terminal domain variants bearing this carboxy-terminal SsrA peptide tag is shown here to depend on the cytoplasmic proteases ClpXP and ClpAP. Degradation in vitro of SsrA-tagged substrates was reproduced with purified components and required a substrate with a wild-type SsrA tail, the presence of both ClpP and either ClpA or ClpX, and ATP. Clp-dependent proteolysis accounts for most degradation of SsrA-tagged amino-domain substrates at 32 degrees C, but additional proteases contribute to the degradation of some of these SsrA-tagged substrates at 39 degrees C. The existence of multiple cytoplasmic proteases that function in SsrA quality-control surveillance suggests that the SsrA tag is designed to serve as a relatively promiscuous signal for proteolysis. Having diverse degradation systems able to recognize this tag may increase degradation capacity, permit degradation of a wide variety of different tagged proteins, or allow SsrA-tagged proteins to be degraded under different growth conditions.

Adenosine Triphosphatases↗

PubMed1998

Biophysical characterization of the TraY protein of Escherichia coli F factor.

The TraY protein is required for efficient bacterial conjugation by Escherichia coli F factor. TraY has two functional roles: participating in the "relaxosome," a protein-DNA complex that nicks one strand of the F factor plasmid, and up-regulating transcription from the traYI promoter. The traY gene was cloned, and the TraY protein was expressed, purified, and characterized. TraY has a mixed alpha-helix and beta-sheet secondary structure as judged by its circular dichroism spectrum, is monomeric, and undergoes reversible urea denaturation with delta Gu = 6 kcal/mol at 25 degrees C. The kinetics of protein unfolding and refolding, as measured by changes in fluorescence, are complex, suggesting the presence of intermediates or of heterogeneity in the folding reaction. TraY has been classified as a member of the ribbon-helix-helix family of transcription factors but is unusual in appearing to have tandem repeats of the beta alpha alpha motif in the same polypeptide chain. The data presented here show that folding and assembly of the functional (beta alpha alpha)2 unit occurs as an intramolecular reaction and not by cross-folding between different polypeptide chains.

Amino Acid Sequence↗

PubMed1998

Tolerance of a protein helix to multiple alanine and valine substitutions.

BACKGROUND: Protein stability is influenced by the intrinsic secondary structure propensities of the amino acids and by tertiary interactions, but which of these factors dominates is not known in most cases. We have used combinatorial mutagenesis to examine the effects of substituting a good helix-forming residue (alanine) and a poor helix-forming residue (valine) at many positions in an alpha helix of a native protein. This has allowed us to average over many molecular environments and assess to what extent the results reflect intrinsic helical propensities or are masked by tertiary effects. RESULTS: Alanine or valine residues were combinatorially substituted at 12 positions in alpha-helix lambda repressor. Functional proteins were selected and sequenced to determine the degree to which each residue type was tolerated. On average, valine substitutions were accommodated slightly less well than alanine substitutions. On a positional basis, however, valine was tolerated as well as alanine at the majority of sites. In fact, alanine was preferred over valine statistically significantly only at four sites. Studies of mutant protein and peptide stabilities suggest that tertiary interactions mask the intrinsic secondary structure propensity differences at most of the remaining residue positions in this alpha helix. CONCLUSIONS: At the majority of positions in alpha-helix lambda repressor, tertiary interactions with other parts of the protein can be viewed as an environmental "buffer" that help to diminish the helix destabilizing effects of valine mutations and allow these mutations to be tolerated at frequencies similar to alanine mutations.

Alanine↗