High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry

Abstract

We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS(3)). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the beta-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1beta, to build an FGF-2 homology model with a backbone error of 4.8 A (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation.

Figure 1

Experimental Design. (Top) Cross-linking of FGF-2. (Middle) Purification of monomeric FGF-2. (Bottom) Proteolytic digestion, HPLC separation, and mass spectrometry.

Figure 2

Mass spectrometry. (A) MALDI-TOF spectrum from tryptic digest of BS³ cross-linked FGF-2. Cross-linked peptides are identified by using the program asap and are denoted with an asterisk (9). (B) MALDI-PSD spectrum of cross-linked peptide E45–R60 (M + H⁺ = m/z 2059.08). (C) MALDI-PSD spectrum of cross-linked peptides L23–R33 and E45–K52 (M + H⁺ = m/z 2465.25). Fragments from peptide L23–R33 are labeled with an α subscript; those from peptide E45–K52 with a β subscript. Fragments due to amide bond cleavages (b-ions, y-ions, and immonium ions) are labeled according to their single letter abbreviations. The most abundant fragments arose from cleavages at cross-linked lysines and aspartic acid. Peaks at m/z 696 and 1771 correspond to fragmentation at y_6β and/or y_6α, and b_2β and/or b_5α, respectively.

Figure 3

The 15 nonlocal throughspace distance constraints generated by the chemical cross-links (yellow dashed lines) superimposed on the average NMR structure of FGF-2 (1BLA). The 14 lysines of FGF-2 are shown in red.

Figure 4

FGF-2 homology model based on IL-1β (cyan) aligned to the NMR structure (white) of FGF-2. Insertions of FGF-2 relative to IL-1β were not modeled. Structural gaps were closed with 100 steps of energy minimization by using Tripos (St. Louis) sybyl 6.4. The backbone rms deviation is 4.8 Å over 101 residues.