Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Glycosyl transferases in mouse and human milk fat globule membranes

Summary Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet     

S-adenosylmethionine and morphogenesis inMucor racemosus

The intracellular concentration of S-adenosylmethionine (SAM) and the specific activity of S-adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) were examined in wild-typeMucor racemosus, as well as a morphological mutant termedcoy, under conditions designed to prevent the morphogenesis of yeasts to hyphae. When the mutant was grown in a defined medium supplemented with methionine and induced to shift by exposure to air, there was an increase in intracellular SAM analogous to that previously reported with the wild type. However, when thecoy mutant was grown in the absence of methionine, the intracellular concentration decreased dramatically and the mutant failed to undergo the yeast to hypha transition. An inhibitor of SAM synthetase activity, cycloserine, was used to lower the intracellular concentration of SAM in the wild-type organisms. Under these conditions, wild-typeM. racemosus failed to undergo the transition from yeasts to hyphae when exposed to air.     

Role of adrenergic innervation in experimental renal hypertension

Renal hypertension was induced by ligation of the aorta between renal arteries in rats sympathectomized with 6-hydroxydopamine. In the early phase, equally severe hypertension developed in the denervated group as compared to innervated controls. Later, blood pressure was lower in the denervated rats. Initially, increases in plasma renin were seen in both groups; the levels, however, were markedly lower in the denervated rats. Later, the renin levels were similar and not different from baseline. It is concluded that adrenergic neural activity is not essential in the development of renal hypertension; the maintenance of the chronic state, however, depends in part on adrenergic innervation.     

Differences in blastema-associated proteins according to position along body axis in regenerating planarians

To test the hypothesis that neoblasts in different positions in regenerating pieces of planarians may be differentially responsive to diffusible stimuli through differentially expressed membrane receptors, we compared membrane surface proteins in blastemas induced at various positions along an anterior-posterior axis in Dugesia gonocephala (Dugès). The proteins were biotinylated and identified by molecular weight in SDS-PAGE (sodium-dodecylsulfate polyacrilamide gel electrophoresis). This SDS-PAGE pattern was then compared with that of N-glycosylated proteins incorporating ³H-mannose. One 20 kDa glycoprotein present in all blastemas at 3 d was absent from more anterior blastemas at 6 d irrespective of whether those blastemas were at the cephalic or the caudal end of the regenerating piece. The expression of this protein appears to be determined by the position of the blastema along the body axis rather than by its prospective fate.     

Transformation of Acidiphilium by electroporation and conjugation.

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.     

Sensory rhodopsin I: Receptor activation and signal relay

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor inHalobacterium halobium. Two aspects are emphasized: (i)The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of anH. halobium mutant lacking all retinylidene proteins known in this species to SR-I⁺ and bacteriorhodopsin (BR)⁺. (ii)The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein (MPP-I) associated with SR-I and found in theH. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g.,Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I MPP-I signal relay.