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Journal Issue - Volume 9 Issue 5 (2000)

Abstract The solution structure and stability of N‐terminally truncated β2‐microglobulin (δN6β2‐m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that δN6β2‐m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full‐length proteins at μM concentrations at pH...

Abstract The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35 ± 0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double‐wound Greek‐key fold with two α‐helices toward...

Abstract The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated....

Abstract Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high‐resolution solid‐state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local α‐helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a...

Abstract The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X‐ray crystallographic studies. The S45A mutant exhibits a 1,700‐fold greater disso‐ciation rate and 907‐fold lower equilibrium affinity for biotin relative to wild‐type streptavidin at 37dGC, indicating a crucial role in binding energetics. The crystal structure of the biotin‐bound...

Abstract An amino‐terminal fragment of human apolipoprotein E3 (residues 1‐165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P212121) crystal form of the amino‐terminal 22 kDa fragment of apolipoprotein E (residues 1‐191). A second orthorhombic crystal form (P212121 with...

Abstract Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N‐terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the...

Abstract Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R‐values...

Abstract Using a combination of theoretical sequence structure recognition predictions and experimental disulfide bond assignments, a three‐dimensional (3D) model of human interleukin‐7 (hIL‐7) was constructed that predicts atypical surface chemistry in helix D that is important for receptor activation. A 3D model of hIL‐7 was built using the X‐ray crystal structure of interleukin‐4 (IL‐4) as a template (Walter MR et al., 1992, J...

Abstract The heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease‐inhibitor complex; thus, the elastase‐ovomucoid system provides a unique opportunity to study phosphate‐protein interactions. We have used...

Abstract The native form of inhibitory serpins (serine protease inhibitors) is not in the thermodynamically most stable state but in a metastable state, which is critical to inhibitory functions. To understand structural basis and functional roles of the native metastability of inhibitory serpins, we have been characterizing stabilizing mutations of human α1‐antitrypsin, a prototype inhibitory serpin. One of the sites that has been shown to be...

Abstract Helical coiled‐coils and bundles are some of the most common structural motifs found in proteins. Design and synthesis of α‐helical motifs may provide interesting scaffolds that can be useful as host structures to display functional sites, thus allowing the engineering of novel functional miniproteins. We have synthesized a 38‐amino acid peptide, α2p8, encompassing the α‐helical hairpin present in the structure of p8MTCP1, as an...

Abstract One alternative method for drug delivery involves the use of siderophore‐antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron‐chelating siderophores, can be actively transported across the outer membrane of Gram‐negative bacteria. These “Trojan Horse” antibiotics may prove useful as an efficient means to combat multi‐drug–resistant bacterial...

Abstract Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium‐binding regulatory protein, has been cleaved by thrombin to create two fragments, TM1 (1‐106) and TM2 (107‐148). NMR and CD results indicate that TM1 and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix‐loop‐helix domains, but rather within an α‐helix....

Abstract The three‐dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI‐1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular β‐sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by...

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