ANALYSIS OF PROTEIN-LIGAND COMPLEX

Analysis of Protein-Ligand Complex



Analysis of Protein-Ligand Complex

Introduction

Experimental assessment of protein stability has previously been employed to identify ligands and optimum buffer conditions by a variety of methods. Differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) are 2 methods that recently have been used to screen soluble proteins against libraries of compounds in the 384-well format. However, the presence of detergent micelles adds complexity to the preparation of membrane proteins, limiting the application of the fluorescence based methods such as DSF, causing high background and poor signal-to-noise ratios. Some attempts have been made to measure the stability of membrane proteins using DSF (Vedadi, Niesen, Allali-Hassani, Fedorov, Finerty, Wasney, 2006, 15835-15840). However, hydrophobic dyes such as SY PRO-orange used in DSF partition into detergent micelle and protein-detergent complexes and cause considerable increase in fluorescence background. Similarly, interaction of reporter dyes with solvent-accessible hydrophobic areas within some folded soluble or membrane proteins can cause a significant increase in the background.

Low signal-to-noise ratios usually necessitate tedious data manipulation and background subtractions. In addition, in some cases, the presence of a reporter dye may affect stability and folding state of the protein. Chemical reactivity of the cysteine residues embedded in the interior of proteins has also been used to detect protein unfolding. In this method, thiol-specific probes such as CPM (N-[4-(7- diethylamino-4-methyl-3-coumarinyl) phenyl] maleimide) were used to monitor membrane protein unfolding by detecting the exposed thiol groups during protein denaturation.

Discussion and Analysis

Ligand binding

MsbA with a transition temperature of 40 °C was stabilized by 4.5 °C in the presence of 2 mM AT P (Fig. 2A), and CpxA was stabilized by 5 °C in the presence of 10 mM AT P but was not affected by nicotinamide adenine dinucleotide (NA DH), which was not expected to bind (Fig. 2B). Interestingly, CorA was stabilized by the divalent cautions, FIG. 1. Thermo-denaturation of membrane proteins in the presence of detergents; CpxA (?) and CorA (?) at 1 mg/mL in n-dodecyl-ß-dmaltoside (DDM; 2× critical micellar concentration CMC) and MsbA (?) at 0.5 mg/mL in DM (2× CMC) were screened by differential static light scattering (DSLS). The solid line represents the best fit for each curve using the Boltzmann equation. N-decyl-ß-d-maltopyranoside (DM; ¦), DDM (?), n-dodecylphosphocholine (FC12; ?), ANZERGENTR (?), and lauryldimethylamine-N-oxide (LDAO; ?) at 4×CMC did not change the background and were not detected by DSLS.

Mg++ and Ca++, as expected but not Li+, indicating that DSLS can detect specific ligand binding (Fig. 2C); to further evaluate the sensitivity of DSLS in detecting ligands, different ligands were titrated against these proteins. Lipid A, a known substrate for MsbA, stabilized this protein in a concentration-dependent manner with as much as 4 °C at a 100-µM concentration (Niesen, Berglund, Vedadi, 2212-2221) (Fig. 3A). Increase in the stability of wild-type MsbA upon increase in concentration of AT P was also detectable (Fig. 3B). A stabilizing effect of AT P on CpxA was also concentration dependent (Fig. 3C). CorA is known to bind and transport Mg++, and ...