The use of biophysical methods increases success in obtaining liganded crystal structures

Abstract

In attempts to determine the crystal structure of small molecule-protein complexes, a common frustration is the absence of ligand binding once the protein structure has been solved. While the first structure, even with no ligand bound (apo), can be a cause for celebration, the solution of dozens of apo structures can give an unwanted sense of déjà vu. Much time and material is wasted on unsuccessful experiments, which can have a serious impact on productivity and morale. There are many reasons for the lack of observed binding in crystals and this paper highlights some of these. Biophysical methods may be used to confirm and optimize solution conditions to increase the success rate of crystallizing protein-ligand complexes. As there are an overwhelming number of biophysical methods available, some of the factors that need to be considered when choosing the most appropriate technique for a given system are discussed. Finally, a few illustrative examples where biophysical methods have proven helpful in real systems are given.

Figure 1

(a) Schematic diagram of an isothermal titration calorimetry instrument, consisting of a sample cell that contains one binding component, an automated syringe that contains the other binding partner and a reference cell. (b) Differences between the sample-cell and reference-cell temperatures induced by binding are initially translated to the power needed to bring the two samples back to the same temperature, before conversion to a binding enthalpy in molar terms.

Figure 2

(a) Flowchart of a typical protocol to produce a solution of a complex, where protein and ligand are simply combined. (b) The simple combination of protein and ligand may be replaced by an ITC, where additional data on the binding stoichiometry, enthalpy and affinity can be gathered en route to crystallization trials.

Figure 3

(a) The aromatic portion of the spectrum of the collagen-like peptide Ac-(GPO)₂GFOGER(GPO)₃-NH₂ is shown at 277, 288 and 298 K. The far left peak highlighted in this spectrum corresponds to the triple-helical form of the peptide and increases in abundance at lower temperatures. (b) Spectra of the same aromatic region at 298 K at varying protein:ligand (P:L) ratios is shown. The preferential broadening of the triple-helical form on protein addition is highlighted by the arrow.

Figure 4

(a) The bottom panel shows a schematic representation of a two-dimensional ¹H–¹⁵N correlation spectrum. Peaks in this spectrum can be assigned to amide NH pairs in the protein backbone. (b) On addition of a ligand, NH pairs close to the binding molecule will be selectively perturbed, allowing the binding site to be localized.

Figure 5

Overlay of the crystal structures of the Src SH2 domain with acetylated pYEEI peptide (PDB code 1a1b; in yellow) and the urazole derivative of the YEEI peptide (in green; Chung et al., in preparation). The extensive hydrogen-bonding interactions made by the urazole are shown by dotted green lines. The excellent phosphate mimicry of the urazole heterocycle within this recognition pocket is evident.

Figure 6

An idealized plot of the fluorescence changes that occur during the thermal denaturation of two proteins when a fluorescent dye such as ANS or Sypro Orange is used. The left curve shows the trace from a protein that denatures with T _m = 318 K; the right curve shows one with T _m = 333 K.