Adina Niv

On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20.

Type I chaperonins are fundamental protein folding machineries that function in eubacteria, mitochondria and chloroplasts. Eubacteria and mitochondria contain chaperonin systems comprised of homo-oligomeric chaperonin 60 tetradecamers and co-chaperonin 10 heptamers. In contrast, the chloroplast chaperonins are heterooligomeric tetradecamers that are composed of two subunit types, alpha and beta. Additionally, chloroplasts contain two structurally distinct co-chaperonins. One, ch-cpn10, is probably similar to the mitochondrial and bacterial co-chaperonins, and is composed of 10 kDa subunits. The other, termed ch-cpn20 is composed of two cpn10-like domains that are held together by a short linker. While the oligomeric structure of ch-cpn10 remains to be elucidated, it was previously suggested that ch-cpn20 forms tetramers in solution, and that this is the functional oligomer. In the present study, we investigated the properties of purified ch-cpn10 and ch-cpn20. Using bifunctional cross-linking reagents, gel filtration chromatography and analytical ultracentrifugation, we show that ch-cpn10 is a heptamer in solution. In contrast, ch-cpn20 forms multiple oligomers that are in dynamic equilibrium with each other and cover a broad spectrum of molecular weights in a concentration-dependent manner. However, upon association with GroEL, only one type of co-chaperonin-GroEL complex is formed.

Solid-phase assay for d-biotin on avidin-cellulose disks

Abstract Filter-paper disks of uniform size were chemically modified by the introduction of isonitrile functional groups. Avidin was then covalently linked to the disks in a four-component condensation reaction involving disk isonitrile groups and avidin carboxyl groups in the presence of a water-soluble aldehyde and an amine. Quantitative assay of unknown d -biotin solutions could be conveniently carried out with such avidin-cellulose disks by a two-step procedure: (i) immersion in the unknown sample, and (ii) exposure to an excess of radioactive biotin. Based on the known total capacity of the disks for biotin, the amount of unlabeled biotin extracted from solution by avidin-cellulose disks could be easily estimated.

Significance of the N-terminal Domain for the Function of Chloroplast cpn20 Chaperonin

Chaperonins cpn60 and cpn10 are essential proteins involved in cellular protein folding. Plant chloroplasts contain a unique version of the cpn10 co-chaperonin, cpn20, which consists of two homologous cpn10-like domains (N-cpn20 and C-cpn20) that are connected by a short linker region. Although cpn20 seems to function like other single domain cpn10 oligomers, the structure and specific functions of the domains are not understood. We mutated amino acids in the “mobile loop” regions of N-cpn20, C-cpn20 or both: a highly conserved glycine, which was shown to be important for flexibility of the mobile loop, and a leucine residue shown to be involved in binding of co-chaperonin to chaperonin. The mutant proteins were purified and their oligomeric structure validated by gel filtration, native gel electrophoresis, and circular dichroism. Functional assays of protein refolding and inhibition of GroEL ATPase both showed (i) mutation of the conserved glycine reduced the activity of cpn20, whether in N-cpn20 (G32A) or C-cpn20 (G130A). The same mutation in the bacterial cpn10 (GroES G24A) had no effect on activity. (ii) Mutations in the highly conserved leucine of N-cpn20 (L35A) and in the corresponding L27A of GroES resulted in inactive protein. (iii) In contrast, mutant L133A, in which the conserved leucine of C-cpn20 was altered, retained 55% activity. We conclude that the structure of cpn20 is much more sensitive to alterations in the mobile loop than is the structure of GroES. Moreover, only N-cpn20 is necessary for activity of cpn20. However, full and efficient functioning requires both domains.

Isonitrile derivatives of polyacrylamide as supports for the immobilization of biomolecules

Isonitrile derivatives of crosslinked polyacrylamide beads (Biogell P-100) were prepared by a two-step procedure: a. N-hydroxymethylation (methylolation) of amide groups on the polymer by treatment with formaldehyde; and b. Attachment of side chains, containing isonitrile functional groups by a displacement reaction involving 1-tosyloxy-3-isocyanopropane (p-CH3-C6H4·SO2·O·(CH2)3 NC) and alkoxide ions generated on methylolated polyacrylamide by treatment with a strong base in a polar aprotic solvent.The modified polyacrylamide beads were tested as support for the immobilization of proteins, and low mol wt ligands by four component condensation (4CC) reactions.Trypsin-polyacrylamide acting on N-benzoyl-L-arginine ethylester exhibited nonlinear Michaelis Menten kinetics and distorted pH activity profiles. The kinetic anomalies could be reduced by increasing the concentration of buffer. The data were consistent with a model assuming “buffer facilitated proton transport” in a diffusionally constrained system.

Studies on the interaction of a surface-bound ligand with a multi-valent high-molecular-weight ligate : The biotinycellulose—avidin system

Abstract Two types of biotinylated cellulose disks were examined: filter-paper disks to which biotin had been covalently attached directly to the paper surface (biotinylcellulose) and disks on which biotin was attached to polyacrylamide side-chains grafted onto the filter-paper surface (biotinylpolyacrylamide—cellulose). The amount of avidin taken up from solution by these disks was linearly related to the avidin input concentration and could be estimated by exposure to [ 14 C]biotin. The avidin-binding capacity of the disks depended on the surface density of covalently attached ligand and exhibited hyperbolic, Langmuir-type behaviour for both types of disks. The [ 14 C]biotin binding capacity of avidinylated disks, on the other hand, showed anomalous, biphasic behaviour: at higher ligand densities, a decrease in [ 14 C]biotin binding was observed. The largest anomalies were obtained with biotinylpolyacrylamide—cellulose disks. Calculated ratios of bound [ 14 C]biotin vs. amount of avidin tetramer (B/A 4 ) showed a similar biphasic behaviour. A constant value of B/A 4 = 3 was obtained at low ligand densities, whereas B/A 4 decreased monotonously with increasing ligand density and asymptotically approached B/A 4 = 1. The data could be explained by assuming that at high ligand densities tetrameric avidin interacts with more than one surface-bound biotin residue.

Isonitrile Derivatives of Polyacrylamide as Immobilization Matrices

Polymeric matrices bearing isonitrile functional groups have been shown to serve as versatile supports for the immobilization of proteins and low molecular weight ligands by four-component condensation reactions (4CC) carried out in aqueous buffers at neutral pH.14 This communication describes a method for the preparation of isonitrile derivatives of polyacrylamide beads (Biogel P-100). The modified polyacrylamide beads were used as supports for immobilization of proteins and low molecular weight ligands.