A single beta subunit M2 domain residue controls the picrotoxin sensitivity of alphabeta heteromeric glycine receptor chloride channels

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the alphabeta heteromeric glycine receptor relative to the alpha homomeric receptor. By analogy with structurally related receptors, the beta subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the alpha subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the alpha homomeric receptor. Furthermore, the converse F282T mutation in the beta subunit increased the picrotoxin sensitivity of the alphabeta heteromeric receptor. The P278G mutation in the beta subunit did not affect the picrotoxin sensitivity of the alphabeta heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to alpha subunit T258 is specifically required for picrotoxin sensitivity. Mutations to alpha subunit T258 also profoundly influenced the apparent glycine affinity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting 'competitive' antagonist) binds to this residue.     

Infrequent cavity-forming fluctuations in HPr from Staphylococcus carnosus revealed by pressure- and temperature-dependent tyrosine ring flips

Infrequent structural fluctuations of a globular protein is seldom detected and studied in detail. One tyrosine ring of HPr from Staphylococcus carnosus, an 88-residue phosphocarrier protein with no disulfide bonds, undergoes a very slow ring flip, the pressure and temperature dependence of which is studied in detail using the on-line cell high-pressure nuclear magnetic resonance technique in the pressure range from 3 MPa to 200 MPa and in the temperature range from 257 K to 313 K. The ring of Tyr6 is buried sandwiched between a beta-sheet and alpha-helices (the water-accessible area is less than 0.26 nm2), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates 10(1)-10(5) s(-1) were determined from the line shape analysis of H(delta1, delta2) and H(epsilon1,epsilon2) of Tyr6, giving an activation volume DeltaV++ of 0.044 +/- 0.008 nm3 (27 mL mol(-1)), an activation enthalpy DeltaH++ of 89 +/- 10 kJ mol(-1), and an activation entropy DeltaS++ of 16 +/- 2 JK(-1) mol(-1). The DeltaV++) and DeltaH++ values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of DeltaV(double dagger) of 28 to 51 mL mol(-1) and DeltaH++ of 71 to 155 kJ mol(-1). The fairly common DeltaV++ and DeltaH++ values are considered to represent the extra space or cavity required for the ring flip and the extra energy required to create a cavity, respectively, in the core part of a globular protein. Nearly complete cold denaturation was found to take place at 200 MPa and 257 K independently from the ring reorientation process.     

Mass spectrometric method for detecting carbon 13 enrichment introduced by folate coenzymes in uric acid

Using [2-13C]uric acid as a test material, we developed a mass spectrometric procedure that detects and estimates the difference in 13C enrichment at the positions of carbons 2 and 8 of the purine ring. This method could replace radiochemical methods and could trace the incorporation of carbon fragments into the purine ring from 13C-labeled metabolites in humans.     

The timing and manner of disassembly of the apparatuses for chloroplast and mitochondrial division in the red alga Cyanidioschyzon merolae

The timing and manner of disassembly of the apparatuses for chloroplast division (the plastid-dividing ring; PD ring) and mitochondrial division (the mitochondrion-dividing ring; MD ring) were investigated in the red alga Cyanidioschyzon merolae De Luca, Taddei and Varano. To do this, we synchronized cells both at the final stage of and just after chloroplast and mitochondrial division, and observed the rings in three dimensions by transmission electron microscopy. The inner (beneath the stromal face of the inner envelope) and middle (in the inter-membrane space) PD rings disassembled completely, and disappeared just before completion of chloroplast division. In contrast, the outer PD and MD rings (on the cytoplasmic face of the outer envelope) remained in the cytosol between daughter organelles after chloroplast and mitochondrial division. The outer rings started to disassemble and disappear from their surface just after organelle division, initially clinging to the outer envelopes at both edges before detaching. The results suggest that the two rings inside the chloroplast disappear just before division, and that this does not interfere with completion of division, while the outer PD and MD rings function throughout and complete chloroplast and mitochondrial division. These results, together with previous studies of C. merolae, disclose the entire cycle of change of the PD and MD rings. Received: 19 May 2000 / Accepted: 3 August 2000     

Asymmetric catalytic ene-cyclization approach to 2-fluoro-19-nor-1,25-dihydroxyvitamin D(3) A-ring analog with significant transactivation activity

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)2D3) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for the treatment of cancer and psoriasis. However, little information has been available on the binding conformation of the 1alpha,25(OH)2D3 molecule and its analogs with the vitamin D receptor (VDR). Therefore, we synthesized 2alpha-fluorinated A-ring analogs of 19-nor-1alpha,25(OH)2D3 in order to investigate the VDR-binding conformation of the A-rings on the basis of the (19)F NMR analysis. The 2alpha-fluoro-19-nor-1alpha,25-dihydroxyvitamin D3 A-ring analog thus synthesized via a asymmetric catalytic carbonyl-ene cyclization, shows significant activity in transactivation.     

Amide proton temperature coefficients as hydrogen bond indicators in proteins

Correlations between amide proton temperature coefficients (_HN/T) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than –4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between –4 and –1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in -helices are on average 1 ppb/K more negative than in -sheets. In consequence, a number of amide protons in -helices involved in hydrogen bonds shorter than 2 Å show _HN/T < –4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 3₁₀ helices and 98% in -turns have temperature coefficients more positive than –4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than –2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures.     

Mutation in collagen gene induces cardiomyopathy in transgenic mice

In many remodeling tissues, such as the heart, collagen degradation to provide new integrin-binding sites is required for survival. However, complete loss of integrin signaling due to disconnection from extracellular matrix (ECM) leads to apoptosis and dilatation. To test the hypothesis that a mutation in type I collagen gene induces cardiomyopathy, we employed a metalloproteinase-resistant collagen mutant homozygous transgenic male (B6,129-Colla-1) and compared with age-sex matched wildtype C57BL/J6 control mice. At the age of 38-42 weeks, aortic and left ventricle (LV) pressure were measured. The LV wall thickness and diameter were measured by a digital micrometer. The levels of matrix metalloproteinase-2 (MMP-2) activity and cardiospecific tissue inhibitor of metalloproteinase-4 (TIMP-4) were measured by zymography and Western blot analyses, respectively. The levels of collagenolysis were measured by Western blot using anti-collagen antibody. In transgenic and wildtype mice, end-diastolic pressure (EDP) was 8.3 +/- 1.7 and 6.5 +/- 1.1 mmHg; LV diameter was 3.43 +/- 0.07 and 2.94 +/- 0.05 mm; wall thickness was 1.18 +/- 0.03 and 1.28 +/- 0.04 mm; end-diastolic wall stress was 600 +/- 158 and 347 +/- 49 dynes/cm(2), respectively. The increase in LV wall stress was associated with increased MMP-2 activity, increased collagenolysis, and decreased levels of TIMP-4. This leads to reduced elastic compliance in collagen mutant transgenic mice. The occurrence of cardiomyopathy in adult Colla-1 mice may be a significant confounding factor as it may be indicative of increased basal levels of ECM disruption. This phenotype is what would be expected if collagen degradation normally supplies integrin ligands during cardiac muscle remodeling.     

The tubulin ancestor,FtsZ, draughtsman,designer and driving force for bacterial cytokinesis

We discuss in this review the regulation of synthesis and action of FtsZ, its structure in relation to tubulin and microtubules, and the mechanism of polymerization and disassembly (contraction) of FtsZ rings from a specific nucleation site (NS) at mid cell. These topics are considered in the light of recent immunocytological studies, high resolution structures of some division proteins and results indicating how bacteria may measure their mid cell point.