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Journal Issue - Volume 15 Issue 3 (March 2006)

Abstract Caspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK‐CARD and caspase‐1‐CARD (CP1‐CARD), the proteins unfold by an apparent two‐state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK‐CARD, the time constants of the...

Abstract The goal of this work was to determine the binding properties and location of 4′,6‐diamidino‐2‐phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2 ± 0.4 μM for the DAPI–tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C‐terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments,...

Abstract The structural properties of the Aβ42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's disease, were investigated through a combination of ion‐mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the −3 charge state of the peptide, its preferred state under...

Abstract We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross‐linking in the solid state. Protein complexes are freeze‐dried under vacuum, and cross‐links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross‐linked...

Abstract Bacterial chaperonin, GroEL, together with its co‐chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In...

Abstract One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of...

Abstract The thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X‐ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ∼2 × 107 times lower than that of wild‐type streptavidin at 25°C. The corresponding reduction in binding free energy (ΔΔG) of 10.1 kcal/mol was nearly completely due to binding...

Abstract Maltohexaose‐producing amylase (G6‐amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short‐chain amylose (DP = 17). Our previous crystallographic study showed that G6‐amylase has nine subsites, from −6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6‐amylase has very low levels of hydrolytic activities for...

Abstract The techniques of phage‐displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein–protein interactions. Focusing these tools on the interaction between the catalytic sub‐unit of protein kinase A (PKAcat) and caveolin‐1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog...

Abstract The N‐terminal 16‐kDa domain of Escherichia coli Ada protein (N‐Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence‐specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 103‐fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl...

Abstract The GLUT4‐regulating protein, TUG, functions to retain GLUT4‐containing membrane vesicles intracellularly and, in response to insulin stimulation, releases these vesicles to the cellular exocytic machinery for translocation to the plasma membrane. As part of our on going effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an...

Abstract G protein‐coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways triggered by hormones, odorants, peptides, proteins, and other types of ligands. The superfamily is so diverse that many members lack sequence similarity, although they all span the cell membrane seven times with an extracellular N and a cytosolic C terminus. We analyzed a divergent set of GPCRs and...

Abstract The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co‐chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop...

Abstract Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric λ repressor and predominantly...

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