Circular Dichroism Studies of the Binding of o‐Nitrophenyl‐β‐d‐fucoside and o‐Nitrophenyl‐β‐d‐galactoside to Lac Repressor

Abstract

The binding of o-nitrophenyl-P-D-fucoside and o-nitrophenyl-/?-D-galactoside to Escherichia coli lac repressor was investigated by circular dichroism in the wavelength range 300 -400 nm corresponding to the o-nitrophenyl chromophores. The CD signal of both ligands drastically changed when they bound to lac repressor due to the asymmetric interaction of the o-nitrophenyl ring with chemical groups of protein. The CD spectra of bound ligands indicate close similarity between the environment of o-nitrophenyl-P-D-fucoside and o-nitrophenyl-/?-D-galactoside on lac repressor. The CD signal is used to calculate the binding parameters ( K and n) to lac repressor. It is demonstrated that the limited proteolytic digestion of lac repressor which gives a 'core protein' does not affect the environment of both ligands on the protein.A variety of sugars, natural or synthetic, are able to induce the lac operon in vivo [l]. In contrast, many non-metabolized sugars are known to prevent induction in vivo and the best characterized of these anti-inducers is o-nitrophenyl-fi-D-fucoside [2]. In vitro it has been shown by several techniques that these effectors bind to the lac repressor and that inducers strongly decrease the lac repressor-operator interaction, while in contrast anti-inducers enhance the lac repressor-operator interaction [3 -81. Both types of molecules have no effect on lac repressor-nonoperator DNA interaction (Maurizot, unpublished results). Furthermore kinetic measurements have shown that these ligands act by altering the rate of dissociation of the repressor . operator complex, whereas the rate of association is practically unaffected [7,9]. Recently, the occurrence of a new type of ligand was reported [9]. This neutral ligand, onitrophenyl-8-D-galactoside binds to the lac repressor protein, competes with the inducer isopropyl-P-Dthiogalactoside, but does not affect the interaction of lac repressor with operator DNA. Its binding constant to lac repressor determined by competition experiments is of the same order of magnitude as that of the anti-inducer o-nitrophenyl-P-D-fucoside (9.2 x 103 M-' versus 7.1 x lo3 M-l) [9].We have used the fact that both o-nitrophenyl-/?-D-fucoside and o-nitrophenyl-P-D-galactoside bear a chromophore with an absorption band in the wavelength range 300-400 nm (where the lac repressor does not absorb) to study the interaction between these compounds and the protein, using circular dichroism measurements. It is well recognized that the binding of low-molecular-weight chromophoric substances to macromolecules may generate optical activity [lo-121. In the present case the sugars are by themselves optically active; nevertheless the circular dichroism spectra of the liganded sugars provide an important experimental parameter for analyzing the non-covalent interactions between the effectors and the protein. EXPERIMENTAL PROCEDUREE. coli lac repressor from strain BMH 493 (a gift of Prof. Muller-Hill), was purified by ammonium sulfate fractionation and column chromatography on phosphocellulose ...

Thermal denaturation of the core protein of lac repressor

“…This latter discrepancy would be removed if on average only three or less molecules of ONPF are lost per tetramer molecule during denaturation as a result of either fewer molecules being bound before denaturation or significant binding of the ligand by the denatured protein. The fact that the binding of ONPF to repressor is 100 times weaker than that of IPTG at 25 °C (Maurizot & Charlier, 1977) suggests that the former possibility is the more likely.…”

Circular Dichroism and Its Empirical Application to Biopolymers

“…One example of such a study is the binding of o-nitrophenyl-P-Dfucoside and o-nitrophenyl-P-D-galactoside to lac repressor (Maurizot and Charlier, 1977). The asymmetric sugar of both of these molecules induces a small CD in the o-nitrophenyl chromophore, as shown in Fig.…”

“…The CD of o-nitrophenyl-P-D-fucoside (a) and o-nitrophenyl-p-D-galactoside(1)) (redrawn fromMaurizot and Charlier, 1977). These compounds are shown without lac repressor(----) and as a 1 : 1 mixture with lac repressor ( -).…”

Lac repressor binding to single‐stranded polyadenylic acid

“…The fluorescence spectra of the lac repressor in Addition of small amounts of trypsin to lac repressor leads to the formation of a proteolytic core which has lost its binding properties on Zac operator and on non-specific DNA, but still binds inducers [17-191 and anti-inducers [20] . This core protein lacks about 60 amino acids at the N-terminal part of the molecule.…”