title: Two domains of RD3 antifreeze protein diffuse independently.
abstract: Antifreeze proteins (AFPs) make up a class of structurally diverse proteins that help to protect many organisms from freezing temperatures by inhibiting ice crystal growth at temperatures below the colligative freezing point. AFPs are typically small proteins with a relatively flat, slightly hydrophobic binding region that matches the lattice structure of a specific ice crystal plane. The only known two-domain AFP is RD3 from the Antarctic eel pout. It consists of two nearly identical type III domains connected by a nine-residue linker. This protein exhibits higher activity than the single-domain protein at low concentrations. The initial solution structure of RD3 revealed that the domains were aligned so that the binding regions were nearly coplanar, effectively doubling the surface area for binding. A more recent report suggests that the domains may not be aligned in solution but rather diffuse independently. To resolve the issue, we have measured the NMR residual dipolar couplings using alignment media of stretched gels and filamentous phage to determine the relative orientation of the domains. We find that the two domains of RD3 are free to move relative to each other, within the constraint of the flexible nine-residue linker. Our data show that there is no strongly preferred alignment in solution. Furthermore, the flexibility and length of the linker are sufficient to allow the two domains to have their binding faces in the same orientation and coplanar for simultaneous binding to an ice crystal surface.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
authors.forename: Nolan B
authors.initials: NB
authors.lastname: Holland
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Nishimiya
authors.forename: Sakae
authors.initials: S
authors.lastname: Tsuda
authors.forename: Frank D
authors.initials: FD
authors.lastname: Sönnichsen
mesh_terms: D021343:Antifreeze Proteins, Type III / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003198:Computer Simulation; D004058:Diffusion; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D021343:Antifreeze Proteins, Type III; C092813:RD3 protein, eel