Preparation of Sample

3.3.1. Identification of Optimal Set of Ligands to Screen for Binding to TetC

There are several strategies, including random screening of a suitable chemical database, that can be employed to identify sets of potential binders. In this study, two crystal structures of TetC are available (PDB access codes 1AF9 and 1A8D [47,48], http://www.rcsb.org/pdb/), so we employed a structure-based approach.

3.3.1.1. Computational Methods to Identify Suitable Ligands

The first step involved using the crystal structure of the protein is to identify binding sites and to carry out a virtual screen of the ACD. Two surface binding sites, Site-1 and Site-2, are identified by calculating the solvent-accessible surface and using the SPHGEN routine from DOCK 4.01 (49), which packs clusters of spheres into structural pockets. A sphere-atom matching scheme in the DOCK 4.01 program (50-52) is then used to computationally screen the ACD, which currently contains more than 300,000 commercially available compounds, and to predict which molecules will likely bind to Site-1 and Site-2. A variety of structurally diverse ligands are chosen to represent the spectrum of possible candidates, and the best candidates are ranked by energy and contact scores. The top 100 compounds are then visually examined qualitatively to assess the interactions that they form with the site.

3.3.1.2. Further Reduction and Optimization of Set of Ligands

The next step is to scale down further the top 100 compounds obtained from the virtual screen to approx 10 to 20 compounds for each site, using a selection criterion based on cost and availability. The final cut is made by checking the remaining compounds for binding activity by electrospray ionization mass spectroscopy (ESI-MS) (53,54; Table 1). Although this step is not required, it does help to significantly speed up the process in narrowing down the number of likely candidates. The sequential funneling of 300,000 ligands to about a dozen for each site resulted in optimizing the short list of compounds (Fig. 4) to use in our NMR screening studies for binding activity in an aqueous environment. There are several reasons why checking for binding activity in solution is important. In particular, it is a prerequisite for identifying detection reagents that will be used in an aqueous environment. In addition, the synthesis of novel bidendate ligands requires that the individual compounds be compatible with one another and with TetC under similar solvent conditions.

3.3.2. Preparation of Stock Solutions and Sample Solutions

1. Store dry recombinant TetC at -15 to -25°C (see Note 6).

2. Dissolve 1 to 3 mg of dry (lyophilized) TetC protein samples in 500 |L/experiment of 100% D2O to obtain a final concentration between 38 and 72 |M (see Note 7).

3. Centrifuge at highest speed for 5 min to remove any insoluble material before adding ligands.

4. Prepare concentrated stock solutions of ligands in either 100 ||L of D2O or deu-terated DMSO. The goal is to keep the total concentration of DMSO at or below 5% (v/v). The addition of this small amount of DMSO would not be expected to affect the stability of the protein, as previously demonstrated by other NMR studies (16), but it should be checked experimentally for each protein by determining whether the binding activity is still detectable for a known binder when the protein is in a 5% DMSO solution. Three ligands, MP-biocytin, lavendustin A, and NF-galactopyranoside, were dissolved in deuterated DMSO; the remaining ligands (Fig. 4) were all soluble in D2O.

5. Calculate the volume of ligand stock solution required to prepare an approx 0.3 to 1 mM concentration in the final 500- to 600-|L sample volume. Test different molar ratios of TetC:doxorubicin ranging from 1:5 to 1:50 in order to determine the best ratios for use in the experiments (see Note 8).

6. Prepare protein-ligand complexes by one of the following two methods:

a. Dissolve 3 mg of TetC in 1.0 mL of D2O (57.9 |M) and centrifuge for 5 min in an Eppendorf microcentrifuge at highest speed to remove insoluble material prior to adding the ligands.

b. Prepare mixtures of ligands in approx 500-800 ||L of D2O (0.2-1 mM) prior to the addition of 1 to 3 mg of dry TetC (38-72 | M) to a final ratio of TetC:ligand of approx 1:20.

Fig. 4. Structures and molecular weights of ligands used in this study. The marker ligand, doxorubicin, is boxed.

Fig. 4. Structures and molecular weights of ligands used in this study. The marker ligand, doxorubicin, is boxed.

Fig. 5. Crystal structure of TetC with doxorubicin, the marker ligand, computationally docked into Site-1. This figure was made using Molscript (60) and Raster3D (61) programs.

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