Search

Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646(Delta405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-A resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The DeltaF508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2-6 have been determined. The barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94 by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.

It has been shown that in aqueous solution histone H1 and H5 C-terminal fragments and peptide hormones beta-endorphin and ACTH adopt preferably the left-handed helical conformation of the poly-L-proline II type. Scanning microcalorimetry and circular dichroism have been used to show that the linear temperature dependence of CD maximum amplitude and partial heat capacity value are broken in the temperature interval between 50 and 60 degrees C, after which [C]p reaches the constant level. It was proposed to be due to noncooperative disordering of the conformation caused by the destruction of the polypeptide hydration shell.