Sergei B. Ruvinov

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. These peptides are 47 amino acids long and contain four or five pairs of electrostatically attractive g ↔ e′ (i, i′ +5) interhelical heterodimeric interactions. The most stable heterodimer consists of an acidic leucine zipper and a basic leucine zipper that melt as homodimers in the micro (Tm = 28°C) or nanomolar (Tm = 40°C) range, respectively, but heterodimerize with a Tm >90°C, calculated to represent femtamolar affinities. Modifications to this pair of acidic and basic zippers, designed to destabilize homodimerization, resulted in peptides that are unstructured monomers at 4 μM and 6°C but that heterodimerize with a Tm = 74°C or Kd(37) = 1.1 × 10−11 M. A third heterodimerizing pair was designed to have a more neutral isoelectric focusing point (pI) and formed a heterodimer with Tm = 73°C. We can tailor this heterodimerizing system to achieve pharmacokinetics aimed at optimizing targeted killing of cancer cells.

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 Å 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). The 181 amino acid protein shows a novel, bilobal arrangement of two αβ modules. Each lobe consists of two α‐helices on the outer side of the molecule, framing a three‐ or four‐stranded antiparallel β‐sheet in the core of the protein. The active site is formed at the interface of the two β‐sheets in a water‐filled cavity involving residues from two H‐X‐T/S‐X motifs. This previously noticed motif participates in coordination of a sulfate ion. A solvent‐exposed surface loop (residues 100–115) is very likely to play a flap‐like role, opening and closing the active site. Based on the crystal structure and on recent mutagenesis studies of a homologous CPDase from Saccharomyces cerevisiae, we propose an enzymatic mechanism that employs the nucleophilic attack of a water molecule activated by one of the active site histidines.

Overexpression and purification of the separate tryptophan synthase α and β 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. Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts. This crystallization procedure is similar to methods used previously to grow crystals of the S. typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts. The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes. Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column. The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis. The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits. The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits.

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 Hisn fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer–trimer equilibria for each protein with association constants in the range of 0.9 × 1010−1.7 × 1011 M−2. All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer–dimer equilibrium in solution.

Subunit communication in the tryptophan synthase α2β2 complex Effects of β subunit ligands on proteolytic cleavage of a flexible loop in the α subunit

To probe the structural basis for ligand‐mediated communication between the α and β subunits in the tryptophan synthase α2β2 complex, we have determined the effects of ligands of the α and β subunits on proteolysis of a flexible loop in the α subunit. We find that addition of a ligand of the β subunit (l‐serine, d‐tryptophan, or l‐tryptophan) in combination with a ligand of the α subunit (α‐glycerol 3‐phosphate) almost completely prevents the tryptic cleavage of the α subunit loop. Thus, the binding of a ligand to the β‐site affects the conformation of the α subunit 25–30 Å distant.