Showing papers by "Robert T. Sauer published in 1995"

Are buried salt bridges important for protein stability and conformational specificity

Abstract: 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

Cooperatively folded proteins in random sequence libraries.

Abstract: 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 hydophobicity 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.

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

Abstract: 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 Bronsted 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

Minor groove DNA-recognition by alpha-helices.

Abstract: The crystal structure of the PurR-DNA complex reveals how α-helices can be used for minor groove recognition and provides a model for the Lad family of repressors.