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J Takei

Publications and source records attributed to J Takei.

11 recordsLinked to original sources
PubMed2000

Absence of stable intermediates on the folding pathway of barnase.

Barnase is one of the few protein models that has been studied extensively for protein folding. Previous studies led to the conclusion that barnase folds through a very stable submillisecond intermediate ( approximately 3 kcal/mol). The structure of this intermediate was characterized intensively by using a protein engineering approach. This intermediate has now been reexamined with three direct and independent methods. (i) Hydrogen exchange experiments show very small protection factors ( approximately 2) for the putative intermediate, indicating a stability of approximately 0.0 kcal/mol. (ii) Denaturant-dependent unfolding of the putative intermediate is noncooperative and indicates a stability less than 0.0 kcal/mol. (iii) The logarithm of the unfolding rate constant of native barnase vs. denaturant concentrations is not linear. Together with the measured rate ("I" to N), this nonlinear behavior accounts for almost all of the protein stability, leaving only about 0.3 kcal/mol that could be attributed to the rapidly formed intermediate. Other observations previously interpreted to support the presence of an intermediate are now known to have alternative explanations. These results cast doubts on the previous conclusions on the nature of the early folding state in barnase and therefore should have important implications in understanding the early folding events of barnase and other proteins in general.

Bacterial Proteins↗
PubMed1999

Relationship between the native-state hydrogen exchange and the folding pathways of barnase.

The previous native-state hydrogen exchange experiment with barnase failed to detect any partially unfolded intermediate state which was contrary to the experimental results from kinetic deuterium hydrogen exchange pulse labeling and protein engineering studies. This has been taken to suggest that the native-state hydrogen exchange method cannot be used alone as an analytical tool to study the folding pathways of proteins. Here, we revisited the pulse labeling experiment with barnase and detected no stable folding intermediate. This finding allows a reconciliation of the native-state HX data and the folding pathway of barnase. Along with alternative theoretical interpretations for a curved chevron plot of protein folding, these data suggest that further investigation of the nature of the intermediate of barnase is needed.

Bacterial Proteins↗
PubMed1999

Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide-dimerised melittin analogues.

The effects of covalent dimerisation of melittin by disulphide formation in cysteine-substitution analogues, (melittin K23C)2 and (melittin K23Q,Q25C)2, on the kinetics of pore formation in phosphatidylcholine small unilamellar vesicles was measured under low ionic strength conditions. The initial rate of melittin-induced pore formation increased with the square of the peptide concentration, whereas both disulphide-dimerised melittin analogues showed a first-order dependence of pore formation rates on peptide concentration. These results indicate that peptide dimerisation is rate-limiting for pore formation under these conditions. A model for a generalised bilayer perturbation resulting from the self-association of a pair of peptide helices at the membrane surface is proposed which may have implications for a number of biological processes that involve the interaction of helical polypeptides with membranes.

Animals↗
PubMed1999

A general method for relieving substrate inhibition in lactate dehydrogenases.

The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure alpha-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD+ binary complex is not perturbed. (2) The mutation also leads to a large increase in the KM for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme-NADH complex.

Enzyme Inhibitors↗
PubMed1998

Self-association of disulfide-dimerized melittin analogues.

Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.

Amino Acid Sequence↗
PubMed1992

Spinal arachnoid cyst in a newborn infant.

A male infant was born with asphyxia following a prolonged breech delivery at 39 weeks of gestation. He had a chylohemothorax from birth. Soon after birth he exhibited flaccid paraplegia with an absence of deep tendon reflexes in the lower extremities. At 17 days of age, magnetic resonance imaging (MRI) disclosed a hypodense area in the thoracic cord between C7 and Th4 in T1-weighted images, and spinal sonography revealed an echo-free space compressing the spinal cord in the same region observed with MRI. A spinal cyst was removed surgically at 24 days of age. The cyst was confirmed by pathology to be a true arachnoid cyst between C7 and Th4. The cause of this cyst may have been a spinal cord injury during the perinatal period.

Arachnoid Cysts↗
PubMed1990

Disseminated adenovirus (type 19) infection in a neonate. Rapid detection of the infection by immunofluorescence.

A case of fatal disseminated adenovirus infection in a neonate who suffered from severe keratoconjunctivitis and pneumonitis is reported. The diagnosis was made seven days after the onset of illness based on the detection of adenovirus antigen in the smears of the tracheal suction and conjunctival swab by immunofluorescence. Viral antigen was detected in the frozen or formalin-fixed autopsy specimens of the lungs, kidneys, spleen, liver and lymph nodes. Typical crystal arrangement of adenovirus virions was observed in the alveolar epithelial cells by electron microscopy. The isolated virus was identified to be of type 19 by a neutralization test. The IF examination using adenovirus group specific immune reagents on the smears of clinical specimens appears to be useful for rapid diagnosis of viral infections.

Adenoviridae Infections↗