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Journal Issue - Volume 12 Issue 1 (January 2003)

Abstract Protein solution nuclear magnetic resonance (NMR) can be conducted in a slightly anisotropic environment, where the orientational distribution of the proteins is no longer random. In such an environment, the large one‐bond internuclear dipolar interactions no longer average to zero and report on the average orientation of the corresponding vectors relative to the magnetic field. The desired very weak ordering, on the order...

Abstract Most small, single‐domain proteins fold with the uncomplicated, single‐exponential kinetics expected for diffusion on a smooth energy landscape. Despite this energetic smoothness, the folding rates of these two‐state proteins span a remarkable million‐fold range. Here, we review the evidence in favor of a simple, mechanistic description, the topomer search model, which quantitatively accounts for the broad scope of observed...

Abstract High molecular weight glutenin subunits (HMW‐GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold‐recognition techniques, we identified a fold compatible with the N‐terminal domain of HMW‐GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein...

Abstract Fragment complementation has been used to delineate the essential recognition elements for stable folding in Src homology 2 (SH2) domains by using NMR spectroscopy, alanine scanning, and surface plasmon resonance. The unfolded 9‐kD and 5‐kD peptide fragments formed by limited proteolytic digestion of the N‐terminal SH2 domain from the p85α subunit of phosphatidylinositol 3′‐kinase fold into an active native‐like structure...

Abstract Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone 15N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8°C). Two exchange processes with different rate constants could be discriminated despite populations of the excited...

Abstract Pressure‐induced unfolding of a molten globule (MG) was studied in a residue‐specific manner with 1H‐15N two‐dimensional NMR spectroscopy using a variant of human α‐lactalbumin (α‐LA), in which all eight cysteines had been replaced with alanines (all‐Ala α‐LA). The NMR spectrum underwent a series of changes from 30 to 2000 bar at 20°C and from −18°C to 36°C at 2000 bar, showing a highly heterogeneous unfolding pattern according to the...

Abstract A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site‐directed mutagenesis was used to introduce cysteine residues at the N‐terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pKa between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the...

Abstract The crystallographic structure of the Escherichia coli OXA‐1 β‐lactamase has been established at 1.5‐Å resolution and refined to R = 0.18. The 28.2‐kD oxacillinase is a class D serine β‐lactamase that is especially active against the penicillin‐type β‐lactams oxacillin and cloxacillin. In contrast to the structures of OXA‐2, OXA‐10, and OXA‐13 belonging to other subclasses, the OXA‐1 molecule is monomeric rather than dimeric and...

Abstract Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the “binary code” strategy preclude explicit design of...

Abstract Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate‐free) form. The final model has been refined at 2.35 Å...

Abstract Cold‐shock proteins (CSPs) bind to single‐stranded nucleic acids, thereby acting as a “RNA chaperone.” To gain deeper insights into the rather unspecific nature of ssDNA/RNA binding, we characterized the binding interface of CspB from Bacillus subtilis to a 25‐mer of ssDNA (Y‐Box25) using heteronuclear 2D NMR spectroscopy. Seventeen residues, including eight out of nine aromatic amino acids, are directly involved in the Y‐Box25...

Abstract The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of...

Abstract High‐sensitivity isothermal titration calorimetry was used to characterize the binding of the glycohydrolitic enzyme hen egg‐white lysozyme to its natural saccharide inhibitors, chitobiose and chitrotriose. Measurements were done at a pH of 4.7, in the 15°C −45°C temperature range. Using a structural‐energetic parameterization derived previously for lectin‐carbohydrate associations, both binding enthalpies and entropies for...

Abstract The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene‐encoded, cysteine‐rich, and cationic peptide that we termed...

Abstract Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent‐exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main‐chain flexibility and thus can promote local distortional motions and large‐scale unfolding. The mutation accelerates amide...

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