Studies on the folding and structure of escherichia coli ribose binding protein

Abstract

Escherichia coli ribose-binding protein (RBP), present in the periplasmic space, is synthesized in the cytosol as a precursor form, exported into the periplasm and then processed into the mature form. In order to investigate the pathway of folding in detail, the precursor RBP (pRBP) and mature RBP (mRBP) were purified and their folding behaviors were compared using various spectroscopic techniques. It was found that the native pRBP and mRBP have virtually identical spectroscopic properties. In UV difference spectra between native and denatured states, both proteins showed minima at 286, 278, 269, 265, and 260 nm which are originated from the tyrosine and phenylalanine residues. When both proteins were excited at 280 nm, the maximum fluorescence emission occurred at 303 nm and the main effect of the denaturant was a decrease of about 45\% in fluorescence intensity without any shift in the maximum wavelength. Analysis of the CD spectra for the native mRBP and pRBP showed that these two proteins have the same secondary structures (mRBP : 44\% $\alpha$-helix, 20\% $\beta$-sheet, 12\% $\beta$-turn, 24\% random structure ; pRBP : 44\% $\alpha$-helix, 20\% $\beta$-sheet, 11\% $\beta$-turn, 25\% random structure). Equilibrium-unfolding studies using two different probes showed no difference in the stabilities between these two proteins. Refolding kinetics observed by CD at 222 nm indicated that the folding of RBPs occurs in two stages, and mRBP folds faster than pRBP. NaCl had no effect on the unfolding kinetics of the RBPs. Substitution of the Phe-187 residue of the RBP with a Trp residue made the RBPs less stable. The differences of midpoints and free energy of denaturation between tryptophan-substituted RBPs and wild type RBPs were found to be 0.08 M and 1.9 kcal/mol, respectively. It was also observed that D-ribose refolds partially unfolded pRBP and mRBP into native structures and decreases the unfolding rate of the these proteins. The conformational stabilities o...