Activity of a two-domain antifreeze protein is not dependent on linker sequence.
The reported NMR structure of RD3, a naturally occurring two-domain antifreeze protein, suggests that the two nearly identical domains are oriented to allow simultaneous binding of their active regions to the ice surface. It is implied that the nine residues linking the two domains play a role in this alignment, but this has not been established. We have designed and expressed a modified form of RD3 that replaces the nine-residue linker with a generic sequence of one serine and eight glycine residues to test the importance of the linker amino acid sequence. The modified linker is shown to have significantly different characteristics compared to the original linker. Heteronuclear nuclear Overhauser effect experiments show that the new linker residues have more mobility than the linker residues in the native protein. Further, NMR data show that the folding of the C-terminal domain is somewhat perturbed by the altered linker. Finally, distributions of residual dipolar couplings indicate that the two domains tumble and move independently of each other. Nevertheless, the thermal hysteresis activity of the modified protein is indistinguishable from that of native RD3, proving that increased activity of the two-domain antifreeze protein is not dependent on structure of the linker.
Holland
Nolan B
NB
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44115, USA.
Nishimiya
Yoshiyuki
Y
Tsuda
Sakae
S
Sönnichsen
Frank D
FD
D000595:Amino Acid Sequence; D021343:Antifreeze Proteins, Type III / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D021343:Antifreeze Proteins, Type III; D003432:Cross-Linking Reagents; C092813:RD3 protein, eel