Flow‐mediated predator–prey dynamics influence fish populations in a tropical river

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Increasing sequence diversity with flexible backbone protein design: the complete redesign of a protein hydrophobic core

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Highlights? Flexible backbone design has been used to mutate every position in a protein core ? The redesign is hyperthermostable (melting temperature >140°C) ? An NMR structure and an X-ray structure closely match the design model ? Designed backbone perturbations were accurately recapitulated     

Structural insight into interactions between dihydrolipoamide dehydrogenase (E3) and E3 binding protein of human pyruvate dehydrogenase complex

The 9.5 MDa human pyruvate dehydrogenase complex (PDC) utilizes the specific dihydrolipoamide dehydrogenase (E3) binding protein (E3BP) to tether the essential E3 component to the 60-meric core of the complex. Here, we report crystal structures of the binding domain (E3BD) of human E3BP alone and in complex with human E3 at 1.6 angstroms and 2.2 angstroms, respectively. The latter structure shows that residues from E3BD contact E3 across its 2-fold axis, resulting in one E3BD binding site on the E3 homodimer. Negligible conformational changes occur in E3BD upon its high-affinity binding to E3. Modifications of E3BD residues at the center of the E3BD/E3 interface impede E3 binding far more severely than those of residues on the periphery, validating the "hot spot" paradigm for protein interactions. A cluster of disease-causing E3 mutations located near the center of the E3BD/E3 interface prevents the efficient recruitment of these E3 variants by E3BP into the PDC, leading to the dysfunction of the PDC catalytic machine.     

Mutations within the S4-S5 linker alter voltage sensor constraints in hERG K+ channels

Human ether-a-go-go related gene (hERG) channel gating is associated with slow activation, yet the mechanistic basis for this is unclear. Here, we examine the effects of mutation of a unique glycine residue (G546) in the S4-S5 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by ∼−50 mV (with the exception of G546C). With the activation shift taken into account, the time constant of activation was also accelerated, suggesting a stabilization of the closed state by ∼1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the α-helical content of the S4-S5 linker suggest that the presence of G546 in wild-type hERG provides flexibility to the helix. Deactivation gating was affected differentially by the G546 substitutions. G546V induced a pronounced slow component of closing that was voltage-independent. Fluorescence measurements of voltage sensor movement in G546V revealed a slow component of voltage sensor return that was uncoupled from charge movement, suggesting a direct effect of the mutation on voltage sensor movement. These data suggest that G546 plays a critical role in channel gating and that hERG channel closing involves at least two independently modifiable reconfigurations of the voltage sensor.     

The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism

A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction.