Time-dependent Conformational Changes Research Articles

Conformational changes in the H3 . H4 histone complex. Serological and circular dichroism studies.

The H3.H4 complex has been extracted from calf thymus chromatin in either 0.05 M NaOAc, pH 5.0, or in 2.0 M KCl, 0.1 M KPO4, pH 6.7, and both forms have been shown to consist solely of histones H3 and H4 when examined on sodium dodecyl sulfate-polyacrylamide gels. Serological and circular dichroism analyses indicate the existence of structural differences between the two forms. Dilution of the high salt form of the complex into low salt produces a time-dependent conformational change which is related to reduction in the alpha helix content of the complex and which exposes antigenic sites on the complex. The existence of multiple forms of the H3.H4 complex may be related to the dynamic equilibrium of nucleosome structure in vivo.

Allosteric properties of rabbit muscle pyruvate kinase

1. 1. The paper reports that rabbit muscle pyruvate kinase displays kinetic properties typical of a regulatory enzyme when examined at pH 7.4. 2. 2. Time-dependent conformational changes in the enzyme are shown. 3. 3. Reasons are suggested to explain why these properties have escaped attention. 4. 4. It is suggested that the results can be explained by assuming that the substrates of the enzyme and the activator, fructose-1, 6-diphosphate, induce conformational changes in the enzyme, some of which are antagonistic in their effects.

The Role of Calcium in the Reactivation of Reduced Taka-amylase

Abstract Aspergillus oryzae α-amylase (Taka-amylase A) is a protein which contains four disulfide bonds, one free sulfhydryl group, and 1 essential atom of calcium per molecule. Full reduction in the presence of ethylenediaminetetraacetate and 8 m urea results in complete loss of enzymic activity. If Ca++ is present, reoxidation at pH 8.6 leads to full recovery of activity with a parallel decrease of free sulfhydryl groups to about one per molecule of protein. However, reoxidation in the absence of Ca++ leads to an inactive protein which contains about three sulfhydryl groups per molecule. Upon the addition of Ca++, this partially reoxidized product is fully reactivated, and the number of sulfhydryl groups again falls to about one per molecule. Reoxidation of unfolded Taka-amylase in the presence of dehydroascorbic acid also leads to rapid decrease in the number of free sulfhydryl groups to about one per molecule. The resulting material is inactive, but the addition of Ca++ results in almost complete restoration of activity. The disulfide bond-containing peptides formed during the reoxidation of reduced Taka-amylase under these various conditions have been located on two-dimensional tryptic peptide maps and on diagonal performic acid-treated peptic peptide patterns. Partially reoxidized, Ca++-free amylase forms three normally paired disulfide bonds, and the addition of Ca++ results in the appearance of the fourth disulfide bond. Dehydroascorbic acid causes all four normally paired disulfide bonds to form, and subsequent activation with Ca++ does not appear to involve disulfide interchange. The Ca++-induced formation of the one remaining disulfide bond in partially reoxidized Taka-amylase is accompanied by time-dependent changes in protein conformation, as detected by ultraviolet difference, solvent perturbation, and fluorescence spectroscopy. The development of a difference spectrum at 286 mµ following the addition of Ca++ closely parallels the recovery of enzymic activity and the loss of the two excess sulfhydryl groups. In addition, the decrease in the number of solvent-accessible chromophores and the loss of the tyrosine fluorescence peak indicate that Ca++ causes conformational transitions involving major portions of the molecule. We conclude that under normal conditions Ca++ is required for full reoxidation and proper folding of reoxidized Taka-amylase, but that Ca++ does not play a role in directing normal pairing of half-cystine residues.