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Journal Issue - Volume 12 Issue 3 (March 2003)

Abstract A solid‐state NMR approach for simultaneous resonance assignment and three‐dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor “shotgun,” of 15N‐labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is...

Abstract We have studied the displacement of Ca2+by the trivalent lanthanide ions (Yb3+) in a protozoan (Entamoeba histolytica) Ca2+‐binding protein (EhCaBP), by NMR and thermodynamics. We have demonstrated, for the first time, how one can use in a combined fashion the utility of NMR and thermodynamics to have an insight to the relative binding specificities/affinity between Ca2+ and Yb3+. As revealed by the titration experiments, Yb3+ displaces Ca2+ from...

Abstract The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to...

Abstract The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two‐dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine‐rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an α‐helical segment (Phe4–Gln14) and a two‐stranded (Phe19–Asp25 and Gln28–Phe33) antiparallel...

Abstract Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM‐5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to...

Abstract We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain•peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ‐binding motif is exposed, and the product sequence bound specifically by a Eu3+chelate‐labeled GST–PDZ ([Eu3+]GST–PDZ). The...

Abstract Human γD crystallin (HγD‐Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174‐residue primarily β‐sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding HγD‐Crys are implicated in early‐onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have...

Abstract WW domains mediate protein–protein interactions in a number of different cellular functions by recognizing proline‐containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty‐two WW domains...

Abstract Increasing resistance of malaria parasites to conventional antimalarial drugs is an important factor contributing to the persistence of the disease as a major health threat. The ongoing search for novel targets has resulted in identification and expression of several enzymes including cysteine proteases that are implicated in hemoglobin degradation. Falcipain‐2 and falcipain‐3 are considered to be the two principal cysteine...

Abstract The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by 15N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting β‐sheets, such as the RT‐loop for example, display increased mobility on picosecond...

Abstract We examined the hydration of amides of α3D, a simple, designed three‐helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy (∼50%–70%), and their...

Abstract To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site‐directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with...

Abstract We investigated the structural determinants of the stability of a designed β‐hairpin containing a natural hydrophobic cluster from the protein GB1 and a D‐Pro‐Gly turn forming sequence. The results of our simulations shed light on the factors leading to an ordered secondary structure in a model peptide: in particular, the importance of the so‐called diagonal interactions in forming a stable hydrophobic nucleus in the...

Abstract The aim of this study has been to develop a strategy for purifying correctly oxidized denatured major histocompability complex class I (MHC‐I) heavy‐chain molecules, which on dilution, fold efficiently and become functional. Expression of heavy‐chain molecules in bacteria results in the formation of insoluble cellular inclusion bodies, which must be solubilized under denaturing conditions. Their subsequent purification and...

Abstract The Neuropeptide Head Activator (HA), pGlu‐Pro‐Pro‐Gly‐Gly‐Ser‐Lys‐Val‐Ile‐Leu‐Phe (pGlu is pyroglutamic acid), is involved in head‐specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher‐organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high...

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