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Journal Issue - Volume 18 Issue 5 (May 2009)

Mutant R96H is a classic temperature-sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 Å resolution one of the bond angles at the site of substitution appeared to...

To try to resolve the loss of stability in the temperature-sensitive mutant of T4 lysozyme, Arg 96 His, all of the remaining 18 naturally occurring amino acids were substituted at site 96. Also, in response to suggestions that the charged residues Lys85 and Asp89, which are 5-8 Å away, may have important effects, each of these amino acids was replaced with alanine. Crystal structures were determined for many of the variants. With the exception of the tryptophan and valine mutants R96W and...

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It is generally accepted that naturally existing functional domains can serve as building blocks for complex protein structures, and that novel functions can arise from assembly of different combinations of these functional domains. To inform our understanding of protein evolution and explore the modular nature of protein structure, two model enzymes were chosen for study, purT-encoded glycinamide ribonucleotide formyltransferase (PurT) and purK-encoded N5-carboxylaminoimidazole ribonucleotide...

A disulfide-linked nitroxide side chain (R1) used in site-directed spin labeling of proteins often exhibits an EPR spectrum characteristic of a weakly ordered z-axis anisotropic motion at topographically diverse surface sites, including those on helices, loops and edge strands of -sheets. To elucidate the origin of this motion, the first crystal structures of R1 that display simple z-axis anisotropic motion at solvent-exposed helical sites (131 and 151) and a loop site (82) in T4 lysozyme have...

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500-fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to...

Human high-density lipoprotein (HDL) plays a key role in the reverse cholesterol transport pathway that delivers excess cholesterol back to the liver for clearance. In vivo, HDL particles vary in size, shape and biological function. The discoidal HDL is a 140-240 kDa, disk-shaped intermediate of mature HDL. During mature spherical HDL formation, discoidal HDLs play a key role in loading cholesterol ester onto the HDL particles by activating the enzyme, lecithin:cholesterol acyltransferase...

The gram-negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high-level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields...

The organization and assembly of the cellulosome, an extracellular multienzyme complex produced by anaerobic bacteria, is mediated by the high-affinity interaction of cohesin domains from scaffolding proteins with dockerins of cellulosomal enzymes. We have performed molecular dynamics simulations and free energy calculations on both the wild type (WT) and D39N mutant of the C. thermocellum Type I cohesin-dockerin complex in aqueous solution. The D39N mutation has been experimentally...

Self-assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self-assembling proteins, named Nanolego. Nanolego consists of structural elements of a structurally stable symmetrical homo-oligomeric protein and binding elements, which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the...

The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. Chemical synthesis is an attractive approach for studying protein domains because it enables the use of unnatural amino acids for site-specific labeling and detailed structure-function analysis. Here, we demonstrate the first total chemical synthesis of the thrombospondin type 1 repeat domain by native chemical ligation. In...