Activation of trisacryl gels with chloroformates and their use for affinity chromatography and protein immobilization

In this study we describe the activation with chloroformates of Trisacryl-GF-2000, a new synthetic gel support that is stable, hydrophilic, and contains large amounts of hydroxyl groups available for activation. Of all the reagents tested, the activation withN-hydroxysuccinimide-chloroformate andp-nitrophenylchloroformate in organic solvents provides the best activation yield and subsequent coupling. When Trisacryl was activated in acetone with the chloroformates in the presence of 4-dimethylaminopyridine as base and catalyst, up to 30% of the hydroxyl groups, (i.e., 1/repeating unit) could be activated. Amino-containing ligands and proteins could be coupled to these carriers at pH 8 or higher. For better results in affinitychromatographic applications, spacers of ε-amino caproic acid or diaminohexane were introduced. The efficacy of these columns was demonstrated by purification of enzymes, antibodies, and antigens. The performance of these new columns were compared with that of Sepharose columns activated in various ways. In every case, the properties of the Trisacryl support proved superior with particular reference to the purity of the product obtained.     

Molecular Replacement Studies of Cucurmosin from Cucurbita Moschata:Structure Homology with Trichosanthin

1 INTRODUCTION Many plants contain proteins that are capable of inactivating ribosomes and therefore are called ribosome-inactivating proteins or RIPs[1]. RIPs are RNA N-glycosidases that inactivate ribosomes through a site-specific deadenylation of the large ribosomal RNA[2, 3]. RIPs are also capable of inactivating many nonribosomal nucleic acid substrates and can be considered as polynucleotide: adenosine glycosidases[4～6]. There are two types of RIPs: type I, single chain pr…     

Exact rotamer optimization for protein design

Computational methods play a central role in the rational design of novel proteins. The present work describes a new hybrid exact rotamer optimization (HERO) method that builds on previous dead-end elimination algorithms to yield dramatic performance enhancements. Measured on experimentally validated physical models, these improvements make it possible to perform previously intractable designs of entire protein core, surface, or boundary regions. Computational demonstrations include a full core design of the variable domains of the light and heavy chains of catalytic antibody 48G7 FAB with 74 residues and 10(128) conformations, a full core/boundary design of the beta1 domain of protein G with 25 residues and 10(53) conformations, and a full surface design of the beta1 domain of protein G with 27 residues and 10(60) conformations. In addition, a full sequence design of the beta1 domain of protein G is used to demonstrate the strong dependence of algorithm performance on the exact form of the potential function and the fidelity of the rotamer library. These results emphasize that search algorithm performance for protein design can only be meaningfully evaluated on physical models that have been subjected to experimental scrutiny. The new algorithm greatly facilitates ongoing efforts to engineer increasingly complex protein features.     

Purification of recombinant green fluorescent protein by three-phase partitioning

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.     

Selective formation of AAB- and ABC-type heterotrimeric alpha-helical coiled coils

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.     

A comparative study of various computational approaches in calculating the structure of pyridoxal 5'-phosphate (PLP)-dependent beta-lyase protein. The importance of protein environment

Various computational approaches, using molecular mechanics (Amber), semiempirical (AM1), density functional (B3LYP), and various ONIOM methods, have been comparatively investigated for the structure of Escherichia coli NifS CsdB protein. The structure of the entire monomer containing 407 amino acid residues and 579 surrounding water molecules has been optimized. The full geometry optimization in the "active site-only" approach (including only active site atoms) has been found to give the largest root-mean-square (RMS) deviation from the X-ray structure; a much better agreement has been achieved by keeping the atoms leading to the backbones of some amino acids frozen in their positions in the X-ray structure. The best agreement has been attained by including the surrounding protein in the calculations using the two-layer ONIOM (B3LYP:Amber) approach. The results presented in this study conclusively demonstrate the importance of the protein/active-site interaction on the active-site structure of the enzyme. The present theoretical study represents the largest system studied at the ONIOM level to date, containing 7992 atoms, including 84 atoms in the QM region and rest in the MM region.     

Studies on the refolding of the reduced-denatured insulin with size exclusion chromatography

The refolding of the reduced-denatured insulin from bovine pancreas was investigated with the size exclusion chromatography (SEC). It was shown that the reduced-denatured insulin originally denatured with 7.0 mol·L-1 guanidine hydrochloride (GuHCI) or 8.0 mol·L-1 urea could not be refolded with a non-oxidized mobile phase. Although the oxidized and reduced glutathione (GSSG and GSH) were employed in the oxidized mobile phase, the reduced-denatured insulin still could not be renatured. However, in the presence of 2.0 mol·L-1 urea in the oxidized mobile phase employed, the reduced-denatured insulin can be refolded with SEC, and the aggregation of denatured insulin can be diminished by urea. In addition, the disul-fide exchange of reduced-denatured insulin also can be accelerated with GSSG/GSH in the oxidized mobile phase. The three disulfide bridges of insulin were formed correctly and the reduced-unfolded insulin can be renatured completely. The results were further tested with re-versed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC).     

Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

The insect spruce budworm(Choristoneura fumiferana) produces antifreeze protein(AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms,known as CfAFP-501 and CfAFP-337,show that both possess similar left-handed β-helical structure,although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to,and cannot be simply accounted for,by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In or-der to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general,we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs,a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337,strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the anti-freeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implica-tion for antifreeze activity are reported for β-helical antifreeze proteins.     

Synthesis and photochemistry of a new class of photocleavable protein cross-linking reagents

A new series of photocleavable protein cross-linking reagents based on bis(maleimide) derivatives of diaryl disulfides have been synthesised. They have been functionalised with cysteine and transient absorption spectra for the photolysis reaction have been recorded by using the pump-probe technique with a time resolution of 100 femtoseconds. Photolysis of the disulfide bond yields the corresponding thiyl radicals in less than a picosecond. There is a significant amount of geminate recombination, but some of the radicals escape the solvent cage and the quantum yield for photocleavage is 30 % in water.     

Conformational Studies on Polytripeptides as a Model of Collagen

To study the role of glycine residues in stabilizing the collagen triple helix, the glycine residues in the polytripeptide (Pro-Ala-Gly)_n were partially replaced by alanine. The proline content was kept constant. The stability of the helical conformation of these polypeptides was studied by IR- and X-ray measurements in the solid state and by ORD, CD and viscometry in solution. The renaturation was followed as a function of time. All the polytripeptides studied, with the exception of (-Pro-Ala-Ala-)_n attained the polyproline II conformation. However the stability decreased with increasing alanine content. Obviously the molecules of (-Pro-Ala-Gly-) are highly associated by intermolecular hydrogen bonds and one may therefore suppose that a triple-stranded helix aggregation occurs. The results of the refolding process show that the stability of the helices seems to also affect the refolding rate in terms of the optical rotation, Two transitions appeared: the first one is responsible for a rapid reversible change in conformation and the second one for a further slow and irreversible change in the hydrodynamic shape. The latter seems to be due to the partial helical nature, leading to higher chain mobility.