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An X-ray diffraction study has been performed by the powder technique on two crystalline forms of yeast cytochrome b2. Type II (DNA-free) cytochrome b2 crystallizes in the trigonal system, the parameters of the unit cell being a = 168.2 Å and c = 112.0 Å, whereas type I (DNA-containing) cytochrome b2 crystallizes in the tetragonal system with a = 99.1 Å and c = 87.0 Å. Measurement of the crystal density and of the partial specific volume of the protein (found to be 0.754 cm³ g at 25°) enables a precise determination of the molecular weight of cytochrome b2 to be obtained. In this respect, the values 234 6000 - 8000 and 235000 - 10000 were found for type II and type I crystals respectively. These results, although in complete concordance, are quite different from the previously accepted value (approx. 180000 from sedimentation studies based on v = 0.71 cm³ /g). Space-groups and protein contents of the crystals show that this protein is composed of at least two, or even four, identical protomers. [ABSTRACT FROM AUTHOR]

An X-ray diffraction study has been performed by the powder technique on two crystalline forms of yeast cytochrome b2. Type II (DNA-free) cytochrome b2 crystallizes in the trigonal system, the parameters of the unit cell being a = 168.2 Å and c = 112.0 Å, whereas type I (DNA-containing) cytochrome b2 crystallizes in the tetragonal system with a = 99.1 Å and c = 87.0 Å. Measurement of the crystal density and of the partial specific volume of the protein (found to be 0.754 cm³ g at 25°) enables a precise determination of the molecular weight of cytochrome b2 to be obtained. In this respect, the values 234 6000 - 8000 and 235000 - 10000 were found for type II and type I crystals respectively. These results, although in complete concordance, are quite different from the previously accepted value (approx. 180000 from sedimentation studies based on v = 0.71 cm³ /g). Space-groups and protein contents of the crystals show that this protein is composed of at least two, or even four, identical protomers. [ABSTRACT FROM AUTHOR]

The I-ScaI/bi2-maturase of Saccharomyces capensis acts as a specific homing endonuclease promoting intron homing, and as a maturase promoting intron splicing. Using the universal code equivalent of the mitochondrial gene encoding the I-ScaI/bi2-maturase, a number of truncated forms of the synthetic gene were constructed, shortened on either side, as were several mutated alleles of the protein. The shortest translation product that fully retains both activities in vivo corresponds to 228 codons of the C-terminal region of the bi2 intron-encoded protein, whereas proteins resulting from more extensive deletions either at the N-terminus or at the C-terminus (up to 73 and four residues, respectively) were able to complement wholly the lack of endogenous maturase, but all lost the endonuclease activity. Similarly, all introduced mutations completely abolished the I-ScaI activity while some mutant proteins retained substantial splicing function. Immunodetection experiments demonstrated that different cytoplasmically translated forms of the I-ScaI/bi2-maturase protein were imported into mitochondria and correctly processed. They appeared to be tightly associated with mitochondrial membranes. Homology modelling of the I-ScaI/bi2-maturase protein allowed us to relate both enzymatic activities to elements of enzyme structure.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1-191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated alpha-helical structure (42-65%) and alters the tertiary structure of the protein although not as much as when apo E3(1-191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1-191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0-4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the alpha-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1-191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.

Group I intron encoded proteins represent a novel class of site specific double strand endonucleases. The endonuclease activity of this class of proteins has been first demonstrated in vivo for I-Sce I which is encoded by a mitochondrial intron of Saccharomyces cerevisiae. Assays using crude cell extracts have shown that I-Sce I can be used in vitro as a restriction endonuclease potentially useful for recombinant DNA technology owing to its large recognition sequence (18 nucleotides). We report here the purification and the first detailed analysis of the in vitro activity and properties of I-Sce I.

We propose a list of 15 water-soluble globular proteins and 13 forms of detergent-soluble membrane proteins of known Stokes radii for the calibration of high-performance liquid chromatography columns. It is shown that it is advisable to use different sets of standards for these two types of proteins as the detergent-solubilized membrane proteins may behave differently, being excluded or retarded, depending upon the gel support. A smooth, although nonlinear, relationship between Stokes radii and erf-1(1--KD) is observed while a large scatter of points exists if the calibration is expressed as the molecular weight as a function of KD.

This paper describes the rapid purification by high-performance liquid chromatographic techniques of milligram quantities of the 7 S ribonucleoprotein complex (RNP 7 S) and its protein component from tench (Tinca tinca) oocyte extracts. High-performance gel permeation chromatography (with a TSK 3000SW column) was found to be unacceptable because of multiple contaminants which coelute with RNP 7 S. In contrast, semipreparative high-performance DEAE ion-exchange chromatography was found to give an excellent separation of the 7 S complex which could be directly adapted to a preparative scale providing rapid purification (less than 1 h) of milligram quantities of the complex. Agarose electrophoresis followed by specific staining of protein and nucleic acid was found to be a convenient and rapid means of evaluating the purification. Finally, reverse-phase high-performance liquid chromatography was found suitable for the purification of the protein component of the 7 S complex.

Small-angle X-ray scattering experiments were performed on an absolute scale on solutions of methionyl-tRNA synthetase from Escherichia coli in its native and trypsin-modified forms. A light-scattering study was performed on the same solutions to verify monodispersity. The structural parameters for the trypsin-modified enzyme, radius of gyration (2.48 nm), volume (90 nm3), surface/volume (1.5 nm-1) and the distribution of chords can account for an equivalent prolate ellipsoid of revolution having an axial ratio 2.3 and a maximum length of 9 nm, with a creviced surface. The rsults obtained for the native enzyme [i.e. radius of gyration (4.3 nm), volume (244 nm3), distribution of the scattering intensity and distribution of chords] exclude the possibility of a very compact quaternary structure and suggest that the enzyme consists of at least two globular parts, probably the two protomers, linked together by interactions involving a limited region of the structure.