2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and Cystic-Fibrosis

The first nucleotide binding fold (NBF1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant form (delta F508,NBF1) were overexpressed in high yield in Escherichia coli in fusion with the maltose-binding protein (MBP). The rationale for producing the chimerae was to aid in domain purification, solubilization, and crystallization and to examine the effect of protein-protein interactions on the properties of the mutant NBF1. Both the purified wild type and delta F508 mutant fusion proteins fold into functional nucleotide binding domains as determined by using the fluorescent nucleotide analog TNP-ATP (2'-(3')-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate). Moreover, the prominent secondary structural features of the two proteins as assessed by ultraviolet circular dichroism spectropolarimetry are very similar, as is the higher order structure evident in three separate protease digestion patterns. Finally, the stability of the nucleotide binding function of the two proteins is similar as assessed by sensitivity to urea. Gel filtration chromatography and electron and confocal microscopy reveal that both fusion proteins, but not MBP alone, form organized fibers, suggesting that NBF1 self-associates, thus raising the possibility that CFTR may be oligomeric in the plasma membrane. Significantly, in the presence of high salt, these fusion proteins also have a propensity to form microcrystals. Finally, the two separate domains (NBF1 and MBP) constituting the fusion proteins appear to interact quite strongly as both proteins remain associated even after cleavage of their fusion junction. The possible relevance of these novel findings to those approaches that might be taken to elucidate the three-dimensional structural differences between the wild type and delta F508 mutant forms of CFTR, as well as to ameliorate the severity of cystic fibrosis, is discussed.

Topics: Adenosine Triphosphate; ATP-Binding Cassette Transporters; Base Sequence; Binding Sites; Carrier Proteins; Chromatography, Gel; Cloning, Molecular; Crystallization; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Hydrolysis; Maltose-Binding Proteins; Membrane Proteins; Microscopy, Electron; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutation; Nucleotides; Protein Denaturation; Recombinant Fusion Proteins

There are several conflicting reports regarding a defective (Ca2+ + Mg2+)-ATPase in tissues from cystic fibrosis (CF) patients. The work presented in this paper represents an independent assessment of (Ca2+ + Mg2+)-ATPase function in CF and, at the same time, provides a somewhat more detailed analysis of the enzyme from CF patients than has previously existed. We found no significant differences in either the Km value for Ca2+ or the Vm value of the (Ca2+ + Mg2+)-ATPase in membrane preparations from CF patients and control subjects when the red cell membranes were prepared by methods which utilize Tris-glycylglycine-Mg2+ buffers. In contrast, the Vm value of the (Ca2+ + Mg2+)-ATPase in the preparations from CF patients was found to be lower than that from control subjects when the membranes were prepared by a series of washes with EDTA-containing buffers (i.e., 1-10 mmol/1 EDTA). The EDTA treatment, however, did not produce any significant difference in the Km between the two groups. The fluorescent ATP analogue, trinitrocyclohexadienylidine-ATP, appeared to interact with erythrocyte ghosts as evidenced from an enhancement of its fluorescence. This enhancement was greater in control preparations than in samples from CF patients. In addition, the kinetic profiles, with respect to ATP, were quite different between the two enzyme populations. The overall results suggest that the lower rate of ATP hydrolysis observed with membranes from CF patients may reflect an impaired utilization of the substrate by the (Ca2+ + Mg2+)-ATPase from these individuals.

Topics: Adenosine Triphosphate; Adolescent; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Transporting ATPases; Child; Child, Preschool; Cystic Fibrosis; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Fluorescent Dyes; Humans; Kinetics; Spectrometry, Fluorescence