Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.     

Tetracyclines modulate cytosolic Ca2+ responses in the osteoclast associated with “Ca2+ receptor” activation

We report the effects of tetracycline analogues on cytosolic Ca²⁺ transients resulting from application of ionic nickel (Ni²⁺), a potent surrogate agonist of the osteoclast Ca²⁺ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca²⁺] response to an application of 5 mM-[Ni²⁺]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca²⁺]. The results suggest a novel action of tetracyclines on the osteoclast Ca²⁺ receptor.     

A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes

Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.     

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in κ-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a κ-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in κ-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.     

The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centres

The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1?Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.     

The relationship between the fatty acid profiles of the yolk and the embryonic tissue lipids: A comparison between the lesser black backed gull (Larus fuscus) and the pheasant (Phasianus colchicus)

Although substantial information is available regarding the fatty acid composition of lipids of the yolk and of the developing tissues of the chicken embryo, there is little knowledge on this topic for other avian species. The aim of the present study was to compare the yolk and embryonic tissue fatty acid profiles for a species selecting its food in the wild (the lesser black backed gull) with one fed on a standard commercial diet (the commercially reared pheasant). The fatty acid compositions of the yolk lipids were determined, and major differences were observed between the two species. In particular, the phospholipid of the gull yolk was enriched in 20:4n-6 and 22:6n-3 (18.8 and 7.1%, respectively, by weight of total fatty acids) in comparison with the pheasant (4.0 and 4.1%, respectively). The fatty acid compositions of the embryonic tissues were determined using eggs incubated in the laboratory. For the liver and heart, the fatty acid composition of the lipids in the two species reflected the initial yolk composition, with the gull tissue lipids generally containing higher proportions of 20:4n-6 and 22:6n-3 than those of the pheasant. In contrast, the fatty acid profiles of the brain phospholipid were essentially identical in the two species, with 20:4n-6 and 22:6n-3 comprising approximately 9 and 17%, respectively, of total fatty acids in both cases.