Comparing protein-ligand interactions in solution and single crystals by Raman spectroscopy

Abstract

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxybenzoate hydroxylase can assume two conformations: buried in the protein matrix ("in") or essentially solvent-exposed ("out"). By using Raman difference spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environments. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, showing that the environments for the flavin's isoalloxazine ring are not identical in the two phases. Moreover, the Raman-band widths of the flavin modes are narrower for both in and out conformers in the crystals, indicating that the flavin exists in a more limited range of closely related conformational states in the crystal than in solution. In general, the ability to compare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.

Figure 1

Structures of the active sites of the PHBH complexes. Water molecules are shown as red spheres, and putative hydrogen bonds are depicted with dotted lines. (A) Holo-PHBH + p-OHB (in conformation). (B) Holo-PHBH + 2,4-di-OHB (out conformation). Structures were obtained from the Protein Data Bank [PDB ID 1PBE, holo-PHBH + p-OHB (in); PDB ID 1DOD, holo-PHBH + 2,4-di-OHB (out)].

Figure 2

Schematic diagram (not to scale) of a Raman microscope. (Inset) Depiction of collection of Raman data from a single protein crystal in a hanging drop. Both video (①) and spectrographic data (②) can be displayed on the computer screen.

Figure 3

Raman difference spectra for FAD bound to wild-type PHBH. (A) Holo-PHBH + p-OHB (in), single crystal. (B) Holo-PHBH + p-OHB (in), in solution. (C) Holo-PHBH + 2,4-di-OHB (out), in solution. (D) Holo-PHBH + 2,4-di-OHB (out), single crystal.

Figure 4

Unsubtracted Raman spectra of a single crystal of holo-PHBH + 2,4-di-OHB (out). Raman peaks identified as prominent flavin-marker bands are indicated (*). Crystal dimensions are approximately 50 × 50 × 30 μm.

Figure 5

Isoalloxazine ring system of FAD, in which —X represents adenosine diphosphate.