Escherichia coli

The ClpA, ClpB, and ClpC subfamilies of the Clp/HSP100 ATPases contain a conserved N-terminal region of ∼150 residues that consists of two approximate sequence repeats. This sequence from the Escherichia coli ClpA enzyme is shown to encode an independent structural domain (the R domain) that is monomeric and ∼40% ␣-helical. A ClpA fragment lacking the R domain showed ATP-dependent oligomerization, proteinstimulated ATPase activity, and the ability to complex with the ClpP peptidase and mediate degradation of peptide and protein substrates, including casein and ssrA-tagged proteins. Compared with the activities of the wild-type ClpA, however, those of the ClpA fragment missing the R domain were reduced. These results indicate that the R domain is not required for the basic recognition, unfolding, and translocation functions that allow ClpA-ClpP to degrade some protein substrates, but they suggest that it may play a role in modulating these activities.

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For many years, human hemoglobin (Hb) isolated from erythrocytes has been investigated as a potential oxygen delivery therapeutic. Advantages with respect to the need for blood typing were balanced with various undesirable properties of cell-free Hb, including cost, overall oxygen affinity, alterations in cooperativity, and ready dissociation into toxic dimeric species. The use of total gene synthesis has resulted in very high levels of functional human Hb expression in Escherichia coli, but there remains a desire for effecting the crosslinking of the hemoglobin tetramer and providing for ready means for increasing the globular molecular weight. In this communication, we report a novel method for linking alpha chains. By circularly permuting one alpha sequence, the second alpha chain in the Hb tetramer can be linked with glycine residues to form 2 bridges across the central cavity. The second alpha chain thus presents its amino and carboxyl termini on a solvent exposed surface, providing for additional polymerization of oxygen-carrying subunits or attachment of any other peptide-based therapeutic. IntroductionA mechanistic and predictive understanding of how a primary sequence can fold to a single tertiary structure remains undefined. Since a thorough understanding of this process will enable the rational design of unique protein structures, this remains an area of active research. One theory suggests that short continuous regions within a protein develop local interactions early in the folding process to minimize the number of accessible structures and catalyze the formation of a functional protein. Radical perturbations of protein structure by circular permutation is one technique being used in an attempt to explore this issue. In addition, utilization of circular permutation itself can be exploited in the design of new protein structures.A circularly permuted protein is created in 2 steps. First, the original termini are linked to form a circular polypeptide. New termini are then created by cleavage of a peptide bond at a location distant from the original termini. Goldenberg and Creighton engineered the first circularly permuted sequence by chemically condensing the termini of bovine pancreatic trypsin inhibitor (BPTI) and generating new termini by limited proteolysis. 1 This circularly permuted variant was shown to refold, in vitro, to a native functional conformation. This seminal experiment suggested that the location of the termini has little effect on the final 3-dimensional structure of the protein. Luger and coworkers genetically circularly permuted phosphoribosyl anthranilate isomerase. 2 The Escherichia coli translated variant was structurally and functionally similar to the wild-type protein. Several other monomeric proteins have since been circularly permuted through genetic manipulations, all of which maintain a nativelike function. [3][4][5][6][7][8][9][10][11] Single subunits of multimeric proteins can also withstand circularly permuted sequences. For example, circularly permuted catalytic cha...