Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis

The residue Tyr 248 of carboxypeptidase A (CPA) is thought to play a role in catalysis by contributing a proton to the incipient amine anion generated during cleavage of peptide substrates. To test this hypothesis we have modified the rat CPA cDNA by site-directed mutagenesis so that the codon for Tyr 248 is replaced by that for Phe. Here, we report the expression of the cDNAs for proCPA and its Tyr-to-Phe variant in yeast via the alpha-factor system. Following zymogen activation by trypsin, wild-type CPA (CPA-WT) and variant CPA (CPA-Phe 248) were purified to homogeneity and characterized enzymatically. CPA-Phe 248 displays essentially undiminished values for the catalytic constant (kcat) towards various peptide and ester substrates. However, the Michaelis constants (Km values) of peptide substrates and the inhibition constant (Ki) of the potato carboxypeptidase inhibitor are increased 6-fold and 70-fold, respectively. These data suggest that the phenolic hydroxyl of Tyr 248 does not act as the requisite general acid catalyst but participates in ligand binding.     

A primary determinant for lipoxygenase positional specificity

The three mammalian lipoxygenases are named according to the carbon position (5, 12 or 15) at which they catalyse the oxygenation of arachidonic acid; they are implicated in inflammatory disorders, for example 15-lipoxygenase is induced in atherosclerosis and can oxidize low-density lipoprotein to its atherogenic form. To identify what determines this positional specificity, we have exchanged conserved differences in the isoforms of 12- and 15-lipoxygenases. Substitution of methionine with valine at position 418 of human 15-lipoxygenase results in an enzyme that performs 12- and 15-lipoxygenation equally. This effect can be mimicked by incubating wild-type 15-lipoxygenase with a synthetically altered substrate which has its doubly allylic methylene carbons shifted by one carbon relative to arachidonic acid. Other mutations at the neighbouring amino acids 416 and 417 give an enzyme which performs 12- and 15-lipoxygenation in a ratio of 15:1. These results indicate that this region might position the substrate in the active site.     

Cyclotides: macrocyclic peptides with applications in drug design and agriculture

Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins.     

墙角节点声传递机理的探讨

对建筑中墙角节点的场传递进行了理论分析，计算了其声透射系数和隔声量。计算结果和实验测试有良好的一致性，并介绍了节点隔声的测试方法。     

“Continuity and change”: representing mass conservation in fluid mechanics

The evolution of the equation of mass conservation in fluid mechanics is studied. Following early hydraulic approximations, and progress by Daniel and Johann Bernoulli, its first expression as a partial differential equation was achieved by d’Alembert, and soon given definitive form by Euler. Later reworkings by Lagrange, Laplace, Poisson and others advanced the subject, but all based their derivations on the conserved mass of a moving fluid particle. Later, Duhamel and Thomson gave a simpler derivation, by considering mass flow into and out of a fixed portion of space. The later propagation of these derivations in nineteenth-century British textbooks and treatises is also examined, including Maxwell’s on the kinetic theory of gases.     

Global landscape of HIV-human protein complexes

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ～40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.