GRANULOCYTE-COLONY-STIMULATING FACTOR MAINTAINS A THERMALLY STABLE, COMPACT, PARTIALLY FOLDED STRUCTURE AT PH-2

At acidic pH many proteins exist in a partially unfolded form, called the ''A'' state. This is defined as a flexible, expanded structure wit h well-defined, usually native-like secondary structure, but no unique tertiary structure, and showing no cooperativity during thermal-induc ed denaturation. Granulocyte-colony stimulating factor (G-CSF), a four -helix bundle cytokine, maintains both thermal stability and tertiary structure at pH 2.0. We therefore examined the conformation and therma l unfolding of G-CSF at pH 2.0, 4.0 and 7.0 using circular dichroism ( CD) and Fourier transform infrared spectroscopy (FTIR). The secondary structure of the molecule remains highly helical as the pH is lowered from 7.0 to 2.0. The tertiary structure of the protein is slightly dif ferent at each pH value, but even at pH 2.0 G-CSF maintains a regular three-dimensional structure. The structure is hydrodynamically compact at these different pH values, with no increase in Stoke's radius even at pH 2.0. The thermal-induced denaturation of G-CSF was determined b y monitoring changes in the CD or FTIR spectra. At pH 2.0 the temperat ure at which thermal-induced denaturation begins is higher than it is at pH 4.0 or 7.0, the thermal unfolding transition remains cooperative and some alpha-helical structure persists even at 86 degrees C. At pH 4.0 and 7.0, secondary and tertiary structures disappear simultaneous ly during thermal denaturation, whereas at pH 2.0 small changes in the far-UV CD region begin to occur first, followed by the simultaneous c ooperative loss of tertiary structure and much of the remaining second ary structure. The structure of G-CSF at pH 2.0 is thus revealed as co mpact, with a unique, three-dimensional structure, highly helical seco ndary structure, and most importantly, a cooperative thermal unfolding transition. G-CSF at acid pH thus does not adopt the ''A'' state. (C) Munksgaard 1997.