Yannis Papanikolau

De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin.

The purification scheme of chitinase A (ChiA) from S. marcescens has been extensively revised. The pure enzyme crystallizes readily under new crystallization conditions. The ChiA crystal structure has been refined to 1.55 A resolution and the crystal structure of ChiA co-crystallized with the inhibitor allosamidin has been refined to 1.9 A resolution. Allosamidin is located in the deep active-site tunnel of ChiA and interacts with three important residues: Glu315, the proton donor of the catalysis, Asp313, which adopts two conformations in the native structure but is oriented towards Glu315 in the inhibitor complex, and Tyr390, which lies opposite Glu315 in the active-site tunnel.

Equilibrium Heat-induced Denaturation of Chitinase 40 from Streptomyces thermoviolaceus

High‐precision differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to study the thermal unfolding of chitinase 40 (Chi40) from Streptomyces thermoviolaceus. Chi40 belongs to family 18 of glycosyl hydrolase superfamily bearing a catalytic domain with a “TIM barrel”‐like fold, which exhibits deviations from the (β/α)8 fold. The thermal unfolding is reversible at pH = 8.0 and 9.0. The denatured state is characterized by extensive structural changes with respect to the native. The process is characterized by slow relaxation kinetics. Even slower refolding rates are recorded upon cooling. It is shown that the denaturation calorimetric data obtained at slow heating rate (0.17 K/min) are in excellent agreement with equilibrium data obtained by extrapolation of the experimental results to zero scanning rate. Analysis of the DSC results reveals that the experimental data can be successfully fitted using either a nontwo‐state sequential model involving one equilibrium intermediate, or an independent transitions model involving the unfolding of two Chi40 energetic domains to intermediate states. The stability of the native state with respect to the final denatured state is estimated, ΔG = 24.0 kcal/mol at 25°C. The thermal results are in agreement with previous findings from chemical denaturation studies of a wide variety of (β/α)8 barrel proteins, that their unfolding is a nontwo‐state process, always involving at least one unfolding intermediate. Proteins 2006.

Crystallization and preliminary X-ray diffraction studies of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp. TAE123

An NAD(+)-dependent psychrophilic alcohol dehydrogenase (ADH) from the Antarctic psychrophile Moraxella sp. TAE123 has been purified to homogeneity. The enzyme consists of four identical subunits, each containing two Zn ions. Protein crystals suitable for X-ray diffraction were obtained under optimized salting-out crystallization conditions using ammonium sulfate as a precipitating agent. The crystals are hexagonal bipyramids and belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = 136.4, c = 210.7 A. They contain one protein homotetramer in the asymmetric unit. Diffraction data were collected to 2.2 A under cryogenic conditions using synchrotron radiation.

Application of the effects of ionic strength reducing agents in the purification and crystallization of chitinase A

The effects of ionic strength reducing agents may find a large number of applications. Based on these effects, we have redesigned the purification scheme of Chitinase A (ChiA) from Serratia marcescen. This scheme led to reproducibly crystallizable enzyme in both salting-in and salting-out conditions, which are presented here. Herein, we demonstrate some experimental applications of the ionic strength reducing agents theory and, in parallel, provide further evidence of the theorys correctness. Finally, we report a new crystal form produced recently in salting-in crystallization experiments. This form may allow the co-crystallization of ChiA mutants with longer substrates.

Crystallization of the E. coli polyamine-induced protein: a novel procedure based on the concept of ionic strength reducers

Nucleation and growth of macromolecular crystals occur in supersaturated solutions the properties of which depend on numerous parameters that in#uence macromolecular solubility. Detailed knowledge of the e!ects of those parameters is essential for crystallization. The concept of the so-called ‘ionic strength reducersa provides insight into the changes of solubility induced by organic solvents and hydrophilic polymers in aqueous electrolytic solutions. A simple and e

High resolution structural analyses of mutant chitinase A complexes with substrates provide new insight into the mechanism of catalysis.

cient procedure is presented which exploits the properties of ionic strength reducers in the crystallization of proteins. Using this procedure in the crystallization of the E.coli polyamine-induced protein, superior crystals compared to conventional techniques have been obtained. The procedure combines microseeding with dialysis techniques and is applicable to other proteins, particularly in cases where conditions favoring both for nucleation and growth cannot be found, or in cases where excessive nucleation leads to the growth of a large number of very small crystals. ( 2000 Elsevier Science B.V. All rights reserved.