Towards a rational approach for heavy-atom derivative screening in protein crystallography

Abstract

Heavy-atom derivatization is routinely used in protein structure determination and is thus of critical importance in structural biology. In order to replace the current trial-and-error heavy-atom derivative screening with a knowledge-based rational derivative-selection method, the reactivity of more than 40 heavy-atom compounds over a wide range of buffer and pH values was systematically examined using peptides which contained a single reactive amino-acid residue. Met-, Cys- and His-containing peptides were derivatized against Hg, Au and Pt compounds, while Tyr-, Glu-, Asp-, Asn- and Gln-containing peptides were assessed against Pb compounds. A total of 1668 reactive conditions were examined using mass spectrometry and were compiled into heavy-atom reactivity tables (http://sis.niaid.nih.gov/cgi-bin/heavyatom_reactivity.cgi). The results showed that heavy-atom derivatization reactions are highly linked to buffer and pH, with the most accommodating buffer being MES at pH 6. A group of 21 compounds were identified as most successful irrespective of ligand or buffer/pH conditions. To assess the applicability of the peptide heavy-atom reactivity to proteins, lysozyme crystals were derivatized with a list of peptide-reactive compounds that included both known and new compounds for lysozyme derivatization. The results showed highly consistent heavy-atom reactivities between the peptides and lysozyme.

Figure 1

Examples of mass spectra illustrating the four-level scale of heavy-atom reactivity based on the derivative peak height. (a) 10 mM buffer; (b) test case carried out in 50 mM buffer. The His-peptide derivatized by potassium tetrachloroaurate in highly reactive MES buffer at pH 6.0 (+++), moderately reactive sodium acetate buffer at pH 5.0 (++), minimal reactive sodium citrate buffer at pH 5.0 (+) and nonreactive HEPES buffer at pH 8.0 (−).

Figure 2

Percentage of conditions in each buffer that are moderately to highly reactive (++/+++), minimally reactive (+) and nonreactive (−) for each peptide group.

Figure 3

Mass spectra for (a) native lysozyme, (b) potassium tetrachloro­platinate(II)-derivatized lysozyme, (c) lead acetate-derivatized lysozyme and (d) potassium tetracyanoplatinate(II)-derivatized lysozyme.

Figure 4

(a) Difference Fourier (F _o − F _c) maps calculated for lysozyme derivatized with lead acetate (in blue density) and with potassium tetracyanoplatinate(II) (in red density) and contoured at the 3σ level. The structure of lysozyme is shown in ribbon representation, with the residues coordinating heavy atoms shown as ball-and-stick models. PyMOL was used to generate the figure. Difference Patterson maps of the lead acetate (b) and potassium tetracyanoplatinate(II) (c) derivatives of lysozyme calculated for the Harker section w = 0.5 with diffraction data between 50 and 3.8 Å resolution. The sections are contoured with 1σ increments starting at 2σ. The respective self Patterson vectors from the individual heavy-atom-binding sites are indicated in the Harker sections.