DNA-Directed Protein Packing within Single Crystals

Abstract

Designed DNA-DNA interactions are investigated for their ability to modulate protein packing within single crystals of mutant green fluorescent proteins (mGFPs) functionalized with a single DNA strand (mGFP-DNA). We probe the effects of DNA sequence, length, and protein-attachment position on the formation and protein packing of mGFP-DNA crystals. Notably, when complementary mGFP-DNA conjugates are introduced to one another, crystals form with nearly identical packing parameters, regardless of sequence if the number of bases is equivalent. DNA complementarity is essential, because experiments with non-complementary sequences produce crystals with different protein arrangements. Importantly, the DNA length and its position of attachment on the protein markedly influence the formation of and protein packing within single crystals. This work shows how designed DNA interactions can be used to influence the growth and packing in X-ray diffraction quality protein single crystals and is thus an important step forward in protein crystal engineering.

Figure 1.. Design and Parameter Scope of mGFP-DNA Conjugates Studied

(A) Schematic of the DNA interaction between mGFP-DNA conjugates with dimensions for the mGFP, the DNA, and the mGFP-DNA linkage. (B) The design parameters explored include DNA sequence, DNA length, amino acid attachment position, and DNA base attachment position. DNA sequence was varied between self-complementary (scDNA), complementary (cDNA), and non-complementary (ncDNA) (upper left). DNA length was varied between 6 and 18 base pairs (upper right). DNA attachment positions were on the side (residue 148) or edge (residue 176 or 191) of the mGFP β-barrel (lower left). The sites within the DNA for attachment to the proteins were either internal or external (lower right).

Figure 2.. mGFP-DNA Single-Crystal Structures

(A) A model of C148 mGFP (top). Four asymmetric units of the C148 mGFP crystal structure (PDB: 6UHJ) in the space group P2₁2₁2₁ (bottom), which is equivalent to previously reported GFP crystal structures (C148 residues represented in blue). Proteins pack densely in this structure, and C148 is involved in an inter-protein interaction. (B) A model of the C148 mGFP-scDNA-1 design (top). Two asymmetric units of the C148 mGFP-scDNA-1 crystal structure (PDB: 6UHL) in the space group P12₁1 (bottom). ^aC148 mGFP-cDNA-1 and C148 mGFP-cDNA-2 crystallize into nearly identical structures (PDB: 6UHN and 6UHO; Figure S20). In these structures, the DNA does not order past the disulfide mGFP-DNA attachment (inset). Pairs of C148 (blue) orient toward distinct regions of solvent space with a C148-C148 distance of 37 ± 4 Å that is within the theoretical distance for DNA hybridization (27–64 Å). (C) A model of the C148 mGFP-ncDNA-1 design (top). Two asymmetric units from the C148 mGFP-ncDNA-1 crystal structure (PDB: 6UHP) in the space group P12₁1 (bottom), where each C148 (orange) orients toward distinct regions of solvent space with no free path between C148 residues that would permit DNA hybridization (Figure S23).

Figure 3.. Confocal Microscopy Evidence for DNA in C148 mGFP-(s) cDNA and C148 mGFP-ncDNA Crystals

(A–C) Confocal microscopy images of (A) C148 mGFP, (B) C148 mGFP-ncDNA-1, and (C) C148 mGFP-cDNA-1 crystals after soaking the crystals for 30 min in the intercalating dye, TOTO-3. The images are in bright field (left), a green channel (middle, 485 nm excitation and 500–550 nm emission filter), and a far-red channel (right, 640 nm excitation and 663–738 nm emission filter). All scale bars represent 50 μm. (D) The ratio of green to far-red fluorescence signals was compared across multiple crystals. The lower signal ratio in C148 mGFP-ncDNA-1 and C148 mGFP-cDNA-1 crystals compared with C148 mGFP crystals indicates the presence of DNA in C148 mGFP-ncDNA-1 and C148 mGFP-cDNA-1 crystals.

Figure 4.. DNA Design Influences mGFP-DNA Packing

(A) A model of the C148 mGFP-cDNA-3 design (top). Four asymmetric units of the C148 mGFP-cDNA-3 crystal structure (PDB: 6UHQ) in the space group C121 (bottom). Increasing DNA duplex length by 3 bp led to this structure. Pairs of C148 (red and purple) orient toward distinct regions of solvent space with a C148-C148 distance of 41 ± 6 Å that is within the theoretical distance for DNA hybridization (37–75 Å). (B) A model of the C148 mGFP-scDNA-2 design (top). Two asymmetric units of the C148 mGFP-scDNA-2 crystal structure (PDB: 6UHR) in the space group P2₁2₁2₁ (bottom). Changing the location of mGFP-DNA attachment position led to this structure. Pairs of C148 (blue) orient toward distinct regions of solvent space with a C148-C148 distance of 30 ± 6 Å that is within the theoretical distance for DNA hybridization (8–45 Å).