Abstract
We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mm concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.
Highlights
The human p8 protein, hp8, is expressed at very low levels in the healthy pancreas, whereas it is highly expressed during the acute phase of pancreatitis and minor pancreatic injuries [1, 2]
We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8, a nucleoprotein whose expression is affected during acute pancreatitis
Further experiments showed that p8 mRNA expression is not restricted to the pancreatic cells, because p8 mRNA is activated in several tissues in response to a systemic lypopolysaccharide injection [3]
Summary
Materials—Deuterium oxide (99% atom in 2H2O) was obtained from Goss Scientific Instruments Ltd. To discard any artifact caused by unfolded protein from freeze drying, the sample in water was diluted 10 times in deuterated water, and the spectrum was recorded and compared with that obtained previously Both spectra were identical (data not shown). DNA Binding Experiments Measured by Circular Dichroism—The DNA binding experiments were carried out by mixing DNA (Type 1 Sigma from calf thymus) and the corresponding form of hp to give the required final concentration rate (weight/weight of protein/DNA) in phosphate buffer, pH 7, at 25 (Ϯ 0.1) °C, and no salt This pH was chosen to map DNA binding because it is close to physiological conditions, and no changes were observed in the shape of the CD spectra with pH (see “Results”).
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