A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I, V, L, N, A, and K.

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.

Plant annexins form calcium-independent oligomers in solution.

The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence.

Biophysical characterization of cyclic nucleotide phosphodiesterases.

We have compared selected biophysical properties of three phosphodiesterases, from Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli. All of them belong to a recently identified family of cyclic nucleotide phosphodiesterases. Experiments elucidating folding stability, protein fluorescence, oligomerization behavior, and the effects of substrates were conducted, revealing differences between the plant and the yeast protein.

Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.

Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M.

We have designed a heterodimerizing leucine zipper system to target a radionuclide to prelocalized noninternalizing tumor-specific antibodies. The modular nature of the leucine zipper allows us to iteratively use design rules to achieve specific homodimer and heterodimer affinities. We present circular-dichroism thermal denaturation measurements on four pairs of heterodimerizing leucine zippers.

Structure and mechanism of activity of the cyclic phosphodiesterase of Appr>p, a product of the tRNA splicing reaction.

The crystal structure of the cyclic phosphodiesterase (CPDase) from Arabidopsis thaliana, an enzyme involved in the tRNA splicing pathway, was determined at 2.5 A resolution. CPDase hydrolyzes ADP-ribose 1",2"-cyclic phosphate (Appr>p), a product of the tRNA splicing reaction, to the monoester ADP-ribose 1"-phosphate (Appr-1"p).

Conformational stability changes of the amino terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system produced by substituting alanine or glutamate for the active-site histidine 189: implications for phosphorylation effects.

The amino terminal domain of enzyme I (residues 1-258 + Arg; EIN) and full length enzyme I (575 residues; EI) harboring active-site mutations (H189E, expected to have properties of phosphorylated forms, and H189A) have been produced by protein bioengineering. Differential scanning calorimetry (DSC) and temperature-induced changes in ellipticity at 222 nm for monomeric wild-type and mutant EIN proteins indicate two-state unfolding. For EIN proteins in 10 mM K-phosphate (and 100 mM KCl) at pH 7.

Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases.

The gene celF of the cryptic cel operon of Escherichia coli has been cloned, and the encoded 6-phospho-beta-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl.

The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily.

The gene glvA (formerly glv-1) from Bacillus subtilis has been cloned and expressed in Escherichia coli. The purified protein GlvA (449 residues, Mr = 50,513) is a unique 6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase (6-phospho-alpha-glucosidase) that requires both NAD(H) and divalent metal (Mn2+, Fe2+, Co2+, or Ni2+) for activity. 6-Phospho-alpha-glucosidase (EC 3.

Engineering the independent folding of the subtilisin BPN' pro-domain: correlation of pro-domain stability with the rate of subtilisin folding.

The 77-amino acid pro-domain greatly accelerates the in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In this complex the pro-domain has a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helixes. When isolated from subtilisin, however, the pro-domain is 97% unfolded even under optimal folding conditions.

Probing the non-proline cis peptide bond in beta-lactamase from Staphylococcus aureus PC1 by the replacement Asn136 --> Ala.

A non-proline cis peptide is present between Glu166 and Ile167 in the active site of beta-lactamase from Staphylococcus aureus PC1. To examine the role of the interaction between the side chain of Asn136 and the main chain of Glu166, the site-directed mutant N136A was produced. The enzyme shows no measurable hydrolytic activity toward a variety of penicillins or cephalosporins except for the chromogenic cephalosporin, nitrocefin.

Engineering the independent folding of the subtilisin BPN' prodomain: analysis of two-state folding versus protein stability.

In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions. Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded.

Prodomain mutations at the subtilisin interface: correlation of binding energy and the rate of catalyzed folding.

The in vivo folding of subtilisin is dependent on a 77 amino acid prosequence, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. The recent determination of the structure of a complex of the prodomain and a calcium-free subtilisin mutant has suggested how the prodomain may catalyze subtilisin folding [Bryan, P., Wang, L.

Catalysis of a protein folding reaction: mechanistic implications of the 2.0 A structure of the subtilisin-prodomain complex.

Biosynthesis of subtilisin is dependent on a 77 amino acid, N-terminal prodomain, which is autocatalytically processed to create the mature form of the enzyme [Ikemura, H., Takagi, H., & Inouye, M.

Monovalent cations partially repair a conformational defect in a mutant tryptophan synthase alpha 2 beta 2 complex (beta-E109A).

We are using the tryptophan synthase alpha 2 beta 2 complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the beta 2 subunit alone is quite low, but is activated by certain monovalent cations or by association with alpha subunit to form an alpha 2 beta 2 complex. Since monovalent cations and alpha subunit appear to stabilize an active conformation of the beta 2 subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of alpha 2 beta 2 complex having beta 2 subunit glutamic acid 109 replaced by alanine (E109A).

Ligand-mediated changes in the tryptophan synthase indole tunnel probed by nile red fluorescence with wild type, mutant, and chemically modified enzymes.

The bacterial tryptophan synthase alpha 2 beta 2 complex contains an unusual structural feature: an intramolecular tunnel that channels indole from the active site of the alpha subunit to the active site of the beta subunit 25 A away. Here we investigate the role of the tunnel in communication between the alpha and beta subunits using the polarity-sensitive fluorescent probe, Nile Red. Interaction of Nile Red in the nonpolar tunnel near beta subunit residues Cys-170 and Phe-280 is supported by studies with enzymes altered at these positions.

Thermal inactivation of tryptophan synthase. Stabilization by protein-protein interaction and protein-ligand interaction.

This study investigates effects of ligands on thermal inactivation of the tryptophan synthase alpha and beta 2 subunits alone and in the alpha 2 beta 2 complex. Addition of pyridoxal phosphate to the apo-beta 2 subunit increases the temperature of one-half inactivation (Ti) from 52 to 77 degrees C. Ligands that promote association of the alpha and holo-beta 2 subunits markedly stabilize the more temperature-labile alpha subunit in the alpha 2 beta 2 complex from irreversible thermal denaturation.

Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium.

To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively. The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit. We also report new and efficient methods for purifying the individual alpha and beta subunits.

Subunit communication in the tryptophan synthase alpha 2 beta 2 complex. Effects of beta subunit ligands on proteolytic cleavage of a flexible loop in the alpha subunit.

To probe the structural basis for ligand-mediated communication between the alpha and beta subunits in the tryptophan synthase alpha 2 beta 2 complex, we have determined the effects of ligands of the alpha and beta subunits on proteolysis of a flexible loop in the alpha subunit. We find that addition of a ligand of the beta subunit (L-serine, D-tryptophan, or L-tryptophan) in combination with a ligand of the alpha subunit (alpha-glycerol 3-phosphate) almost completely prevents the tryptic cleavage of the alpha subunit loop. Thus, the binding of a ligand to the beta-site affects the conformation of the alpha subunit 25-30 A distant.

Mechanism of mutual activation of the tryptophan synthase alpha and beta subunits. Analysis of the reaction specificity and substrate-induced inactivation of active site and tunnel mutants of the beta subunit.

The origin of reaction and substrate specificity and the control of activity by protein-protein interaction are investigated using the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium. We have compared some spectroscopic and kinetic properties of the wild type beta subunit and five mutant forms of the beta subunit that have altered catalytic properties. These mutant enzymes, which were engineered by site-directed mutagenesis, have single amino acid replacements in either the active site or in the wall of a tunnel that extends from the active site of the alpha subunit to the active site of the beta subunit in the alpha 2 beta 2 complex.

[Factors, determining the effectiveness of tryptophanase interaction with amino acids].

Inhibition of tryptophanase-catalyzed decomposition of S-(o-nitrophenyl)-L-cysteine by a variety of amino acids has been investigated. For amino acids similar to the natural substrate and for those having minimal steric requirements for the side chain, the linear correlation exists between-RTlnKi and side chain hydrophobicity. L-ornithine and L-arginine are anomalously potent inhibitors taking into account low hydrophobicity of their side chains.

Tyrosine phenol-lyase from Citrobacter intermedius. Factors controlling substrate specificity.

L-Amino acids are competitive inhibitors of tyrosine phenol-lyase from Citrobacter intermedius. For non-branched amino acids the correlation exists between -RTlnKi and side-chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains.

[Interaction of tyrosine-phenol-lyase from Citrobacter intermedius with amino acids and their derivatives: factors determining the effectiveness of binding].

The inhibition by L-amino acids and their derivatives of tyrosine phenol-lyase is investigated. Tyramine, alpha-phenylethylamine and tryptamine have no detectable inhibition effect and hence are weakly bonded by an active site. The aromatic amino acid amides are competitive inhibitors but do not manifest an enzymatic isotope exchange of alpha-proton in D2O.