Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

Abstract

Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen/high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins. When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

Fig. 1.

The 10-sec deuteration results are shown for 21 proteins that were analyzed, whose amino acid lengths varied from 76 to 461 residues. Dark regions indicated fast-exchanging amides, and clear regions indicate stretches of no exchange. Regions of four or more fast-exchanging amides are circled. Corresponding boundaries for fast-exchanging amides are displayed in Fig. 5.

Fig. 2.

(A) Ten-second amide hydrogen/deuterium exchange map for TM0449. The horizontal blue bars are the protein's pepsin-generated fragments that had been produced, identified, and used as exchange rate probes in the subsequent 10-sec deuteration study. The number of deuterons that went on to each peptide in 10 sec is indicated by the number of red residues in each peptide. Deuterium labeling was assigned to residue positions within the protein by first optimizing consensus in deuterium content of overlapping peptide probes, followed by further clustering of labeled amides together in the center of unresolved regions (with vertical bars indicating the range of possible location assignments), generating the consensus map (Upper), in which two extensive segments are seen to be deuterium-labeled: segment 1 (Phe-31-Glu-38) and segment 2 (Ser-88–Lys-93). (B) The electron density of the crystal indicates two regions of disordered sequence, corresponding to the segments 1 and 2. (D and C) Detailed electron density maps are shown, in which density is not visualized between the Phe-31–Glu-39 and Ser-88–Ser-95 regions of the TM0449 3D structure (45). DXMS-determined disorder constitutes 6.4% of this protein's sequence.

Fig. 3.

The on-exchange map of TM0505 indicates three internal segments (A, B, and C) of rapidly exchanging amides. The internal segments are mapped onto the crystal structure of the GroES protein homolog of TM0505. The M. tuberculosis GroEL subunit is blue, and the heptamer complex of M. tuberculosis GroES subunits is gray. The homologous locations of rapid exchange sites in the T. maritima protein are red. Disorder constitutes 16.3% of this protein's sequence.

Fig. 4.

TM1171 (A) and TM0160 (B) show substantial C-terminal disorder (circled sequences). Four truncated constructs of each protein were made by eliminating the C-terminal regions (D1–D4). (C) Repeat DXMS analysis demonstrates that deletion constructs of TM0160 preserve the core full-length structure. Full-length TM0160, and its longest truncation (D3), were on-exchanged variously for 10, 100, 1,000, and 10,000 sec at 0°C, were exchange-quenched, and were subjected to comparative DXMS analysis as described above. The resulting comprehensive exchange maps for full-length (Upper) and D3 truncated (Lower) had virtually identical patterns (10-sec exchange time is shown).