Three reasons protein disorder analysis makes more sense in the light of collagen

We have identified that the collagen helix has the potential to be disruptive to analyses of intrinsically disordered proteins. The collagen helix is an extended fibrous structure that is both promiscuous and repetitive. Whilst its sequence is predicted to be disordered, this type of protein structure is not typically considered as intrinsic disorder. Here, we show that collagen‐encoding proteins skew the distribution of exon lengths in genes. We find that previous results, demonstrating that exons encoding disordered regions are more likely to be symmetric, are due to the abundance of the collagen helix. Other related results, showing increased levels of alternative splicing in disorder‐encoding exons, still hold after considering collagen‐containing proteins. Aside from analyses of exons, we find that the set of proteins that contain collagen significantly alters the amino acid composition of regions predicted as disordered. We conclude that research in this area should be conducted in the light of the collagen helix.     

A New Look at the Molecular Mechanism of Thymidylate Synthase and Its Interaction with Nucleotide Analogues

Abstract

The increasing availability of the crystal structures of a variety of complexes of thymidylate synthase¹ (TS) and its mutants with nucleotide and folate analogues, has greatly advanced our understanding of the mechanism of catalysis, and the detailed interaction of the enzyme with inhibitors.     

Enzymatic regulation of calcium current in dialyzed and intact molluscan neurons

Isolated neurons of Helix aspersa were dialyzed and voltage clamped under conditions that isolate the Ca current. The rapid time-dependent run-down, or washout, of Ca current could be slowed by addition of 1 mM EGTA to the dialysis solution. A more effective means of slowing washout was the use of agents that promote protein phosphorylation, such as cAMP, Mg-ATP and the catalytic subunit (CS) of cAMP-dependent protein kinase, along with leupeptin, a tripeptide inhibitor of proteases. In the presence of these agents, no internal EGTA was required to prevent Ca current washout. Thus, during dialysis with 100 microM leupeptin, 7 mM Mg-ATP and 20 micrograms/ml CS, the Ca current remained stable for up to several hours. The rate of Ca-dependent inactivation of the current that occurs during a depolarizing step showed only a small decline during prolonged dialysis. Under these conditions, introduction of 10 microM calmodulin plus 40 micrograms/ml calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation during a depolarizing step. This increase in rate of inactivation, as well as the original inactivation, was eliminated by introduction of EGTA or replacement of external Ca with Ba, results that are consistent with the ion dependency for activation of calcineurin. When internal ATP was replaced with ATP-gamma-S, a hydrolysis-resistant analogue, the rate of Ca current inactivation slowed, providing further evidence that inactivation involves a dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of N-hydroxylation as a prerequisite mechanism of nitric oxide synthase inactivation

L-N5-(1-Hydroxyiminoethyl)-ornithine (L-NHIO) and L-N6-(1-hydroxyiminoethyl)-lysine (L-NHIL) were synthesized and tested as potential intermediates in the mechanism-based inactivation of nitric oxide synthase (NOS) by L-N5-iminoethylornithine (L-NIO) and L-N6-iminoethyllysine (L-NIL). Although these compounds were determined to be competitive inhibitors, mechanism-based inactivation was not observed.     

Phosphorylation-induced transient intrinsic structure in the kinase-inducible domain of CREB facilitates its recognition by the KIX domain of CBP

Phosphorylation at Ser-133 of the kinase inducible domain of CREB (KID) triggers its binding to the KIX domain of CBP via a concomitant coil-to-helix transition. The exact role of this key event is still puzzling: it does not switch between disordered and ordered states, nor its direct interactions fully account for selectivity. Hence, we reasoned that phosphorylation may shift the conformational preferences of KID towards a binding-competent state. To this end we investigated the intrinsic structural properties of the unbound KID in phosphorylated and unphosphorylated forms by simulated annealing and molecular dynamics simulations. Although helical populations show subtle differences, phosphorylation reduces the flexibility of the turn segment connecting the two helices in the complexed structure and induces a transient structural element that corresponds to its bound conformation. It is stabilized by the pSer-133-Arg-131 interaction, which is absent from the unphosphorylated KID. Diminishing this coupling decreases the 3.1 kcal/mol contribution of pSer-133 to the binding free energy (DeltaGbind) of the phosphorylated KID to KIX by 1.1 kcal/mol, as computed in reference to Ser-133. In a binding competent form of the S133E KID mutant, the contribution of Glu-133 to DeltaGbind is by 1.5 kcal/mol smaller than that of pSer, suggesting that altered structural properties due to pSer --> Glu replacement impair the binding affinity. Thus, we propose that phoshorylation contributes to selectivity not merely by the direct interactions of the phosphate group with KIX, but also by promoting the formation of a transient structural element in the highly conserved turn segment.     

A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death

In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.