Determination of the solution structure of the N-domain plus linker of Antarctic eel pout antifreeze protein RD3.
RD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn(1)-Glu(64)) and C-terminal (residues Ser(74)-Glu(134)) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D(65)GTTSPGLK(73)-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule. The present paper shows for the first time the solution structure and preliminary (15)N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy. Forty converged structures of RD3-Nl were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-Nl has a globular form comprising six beta-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain. Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain. This definitive linker is roughly divided into two short strands, -D(65)GTTSP(70)- and -G(71)LK(73)-, which are bent around -T(67)TSPG(71)- at an angle of approximately 60 degrees. This bending motif of the linker may function to orient the two ice-binding sites of the N- and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface. (+info)
NMR analysis of type III antifreeze protein intramolecular dimer. Structural basis for enhanced activity.
The structure of a new antifreeze protein (AFP) variant, RD3, from antarctic eel pout (Rhigophila dearborni) with enhanced activity has been determined for the first time by nuclear magnetic resonance spectroscopy. RD3 comprises a unique translational topology of two homologous type III AFP globular domains, each containing one flat, ice binding plane. The ice binding plane of the N domain is located approximately 3.5 A "behind" that of the C domain. The two ice binding planes are located laterally with an angle of 32 +/- 12 degrees between the planes. These results suggest that the C domain plane of RD3 binds first to the ice  prism plane in the <0001> direction, which induces successive ice binding of the N domain in the <0101> direction. This manner of ice binding caused by the unique structural topology of RD3 is thought to be crucial for the significant enhancement of antifreeze activity, especially at low AFP concentrations. (+info)
Contribution of hydrophobic residues to ice binding by fish type III antifreeze protein.
Type III antifreeze protein (AFP) is a 7-kDa globular protein with a flat ice-binding face centered on Ala 16. Neighboring hydrophilic residues Gln 9, Asn 14, Thr 15, Thr 18 and Gln 44 have been implicated by site-directed mutagenesis in binding to ice. These residues have the potential to form hydrogen bonds with ice, but the tight packing of side chains on the ice-binding face limits the number and strength of possible hydrogen bond interactions. Recent work with alpha-helical AFPs has emphasized the hydrophobicity of their ice-binding sites and suggests that hydrophobic interactions are important for antifreeze activity. To investigate the contribution of hydrophobic interactions between type III AFP and ice, Leu, Ile and Val residues on the rim of the ice-binding face were changed to alanine. Mutant AFPs with single alanine substitutions, L19A, V20A, and V41A, showed a 20% loss in activity. Doubly substituted mutants, L19A/V41A and L10A/I13A, had less than 50% of the activity of the wild type. Thus, side chain substitutions that leave a cavity or undercut the contact surface are almost as deleterious to antifreeze activity as those that lengthen the side chain. These mutations emphasize the importance of maintaining a specific surface contour on the ice-binding face for docking to ice. (+info)
The refined crystal structure of an eel pout type III antifreeze protein RD1 at 0.62-A resolution reveals structural microheterogeneity of protein and solvation.
RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62 A. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the "pretzel fold" includes four short beta-strands and a 3(10) helix. There is a novel internal cavity of 45 A(3) surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized. (+info)
Artificial multimers of the type III antifreeze protein. Effects on thermal hysteresis and ice crystal morphology.
A variant of antifreeze protein (AFP) named RD3 from antarctic eel pout (Lycodichthys dearborni) comprises the type III AFP intramolecular dimer, which is known to exhibit a significant enhancement of thermal hysteresis when compared with the type III AFP monomer (Miura, K., Ohgiya, S., Hoshino, T, Nemoto, N., Suetake, T., Miura, A, Spyracopoulos, L., Kondo, H., and Tsuda, S. (2001) J. Biol. Chem. 276, 1304-1310). Here we genetically synthesized intramolecular dimer, trimer, and tetramer of the type III AFP, for which we utilize the genes encoding the primary sequences of the N-domain, the C-domain, and the 9-residue linker of RD3, and we examined the AFP multimerization effects on thermal hysteresis and ice crystal morphology. Significantly, (i) the thermal hysteresis increases in proportion with the size of the multimers, (ii) a larger size of the multimer exerts the maximum activity at lower concentration, (iii) every multimer changes the morphology of a single ice crystal into a unique shape that is similar but not identical to the ordinary hexagonal bipyramid, and (iv) the size of ice crystal becomes dramatically small with increasing the concentration of the multimer. The thermal hysteresis enhancement of the multimer was detected in both molar and domain bases. These results suggest that a molecule comprising the multiple AFP domains connected in tandem acquires an enhanced affinity for the ice binding. (+info)
Ice nucleation inhibition: mechanism of antifreeze by antifreeze protein.
The effect of antifreeze protein type III (one type of fish antifreeze protein) on ice crystallization was examined quantitatively based on a "micro-sized ice nucleation" technique. It was found for the first time that antifreeze proteins can inhibit the ice nucleation process by adsorbing onto both the surfaces of ice nuclei and dust particles. This leads to an increase of the ice nucleation barrier and the desolvation kink kinetics barrier, respectively. Based on the latest nucleation model, the increases in the ice nucleation barrier and the kink kinetics barrier were measured. This enables us to quantitatively examine the antifreeze mechanism of antifreeze proteins for the first time. (+info)
Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner.
We found that Notched-fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP- and QAE-sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC-pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 degrees C for the QAE1 isoform, 0.11 degrees C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes 'active' when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co-operative manner. (+info)
Solution structure of the antifreeze-like domain of human sialic acid synthase.
The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface. (+info)