Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

Abstract

Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters.

Macromolecular NMR spectroscopy for the non‐spectroscopist

“…For reasons that we will not go into, the nuclei of many isotopes such as 1 H, 13 C, 15 N and 31 P carry magnetic dipoles. These dipoles take up different orientations in a magnetic field, such as the magnet of an NMR spectrometer, and each orientation has a different energy.…”

“…1B). Each signal in this latter spectrum has an intensity and two chemical shifts (one for the 1 H and another for the 15 N nucleus) and the spectrum is plotted 'looking from above', much like a topographic map. For a well-behaved protein, the 15 N-HSQC spectrum will contain one peak for each backbone amide proton (i.e.…”

“…For a well-behaved protein, the 15 N-HSQC spectrum will contain one peak for each backbone amide proton (i.e. one for each peptide bond, except those preceding prolines), a peak for each indole NH of tryptophan residues, and pairs of peaks for the sidechain amide groups of each Asn and Gln residue (for these amide groups, each 15 N nucleus has two attached protons). Under favourable circumstances, signals from the guanidino groups of arginine can also be observed.…”

“…C-glucose and 15 NH 4 Cl]. Of course, a protein cannot always be produced recombinantly in bacteria, and isotopic labels are not as economically incorporated into other expression systems, although there are exceptions [8].…”

“…Armed with a simple 1D 1 H-NMR and 15 N-HSQC spectrum, there are a number of questions that can be readily answered to provide valuable information for the crystallographer, the enzymologist or the protein engineer. Below, we discuss some common questions that NMR can be used to address.…”