Increased flexibility decreases antifreeze protein activity

Abstract

Antifreeze proteins protect several cold-blooded organisms from subzero environments by preventing death from freezing. The Type I antifreeze protein (AFP) isoform from Pseudopleuronectes americanus, named HPLC6, is a 37-residue protein that is a single α-helix. Mutational analysis of the protein showed that its alanine-rich face is important for binding to and inhibiting the growth of macromolecular ice. Almost all structural studies of HPLC6 involve the use of chemically synthesized protein as it requires a native N-terminal aspartate and an amidated C-terminus for full activity. Here, we examine the role of C-terminal amide and C-terminal arginine side chain in the activity, structure, and dynamics of nonamidated Arg37 HPLC6, nonamidated HPLC6 Ala37, amidated HPLC6 Ala37, and fully native HPLC6 using a recombinant bacterial system. The thermal hysteresis (TH) activities of the nonamidated mutants are 35% lower compared with amidated proteins, but analysis of the NMR data and circular dichroism spectra shows that they are all still α-helical. Relaxation data from the two nonamidated mutants indicate that the C-terminal residues are considerably more flexible than the rest of the protein because of the loss of the amide group, whereas the amidated Ala37 mutant has a C-terminus that is as rigid as the wild-type protein and has high TH activity. We propose that an increase in flexibility of the AFP causes it to lose activity because its dynamic nature prevents it from binding strongly to the ice surface.

Figure 1

Activity of rHPLC6 and mutant constructs. (A) Ice crystal morphology of the four proteins in the thermal hysteretic gap. The identity of the protein is indicated below each photograph. The scale bar is the equivalent of 25 μm in length for all photographs. (B) Thermal hysteresis measurements. The activities of the constructs were measured using a nanoliter osmometer as described in the Material and Methods section. Synthetic HPLC6, open square; rHPLC6, open circle; rHPLC6-Ala³⁷-NH₂, open triangle; rHPLC6-Arg³⁷, solid circle; rHPLC6-Ala³⁷, solid triangle. The lines indicate the average trend for each of the proteins. Synthetic HPLC6 and rHPLC6, solid line; rHPLC6-Ala³⁷-NH₂, dotted and dashed line; rHPLC6-Arg³⁷, dashed line; rHPLC6-Ala³⁷, dotted line. The error bars represent the standard deviation of three independent measurements.

Figure 2

Global structure of rHPLC6 and mutants. The CD spectra of the proteins, collected at 3°C. rHPLC6, open circle; rHPLC6-Ala³⁷-NH₂, open triangle; rHPLC6-Arg³⁷, solid circle; rHPLC6-Ala³⁷, solid triangle.

Figure 3

Local structure of rHPLC6, rHPLC6-Ala³⁷-NH₂, rHPLC6-Arg³⁷, and rHPLC6-Ala³⁷. (A) Overlapped ¹⁵N-HSQC spectra of rHPLC6 and mutants at 3°C. The residues of all of the assigned peaks of the wild-type protein are labeled with the residue number and single-letter amino acid code. Peaks of the mutant proteins that have shifted significantly [>0.05 ppm as shown in (B)] are also labeled. rHPLC6, purple; rHPLC6-Ala³⁷-NH₂, blue; rHPLC6-Arg³⁷, green; rHPLC6-Ala³⁷, orange. (B) Chemical shift changes of the mutants compared with the wild-type protein. Weighted changes in backbone ¹H and ¹⁵N chemical shifts of rHPLC6-Ala³⁷-NH₂ (open triangles), rHPCL6-Arg³⁷ (solid circles), and rHPLC6-Ala³⁷ (solid triangles) are plotted on a per residue basis.

Figure 4

Relaxation data of rHPLC6 and mutants. The (A) R₁, (B) R₂, and (C) ¹⁵N-NOE data are plotted on a per residue basis. Error bars represent the error in fitting the relaxation decay curves as described in CCPNMR. rHPLC6, open circle; rHPLC6-Ala³⁷-NH₂, open triangle; rHPLC6-Arg³⁷, solid circle; rHPLC6-Ala³⁷, solid triangle.

Figure 5

Cap structure of Type I AFP. The C-terminal region of Type I AFP from the crystal structure (PDB 1WFB, chain B) is shown in stick representation. Asp⁵ of a nearby symmetry mate is also shown. Intramolecular hydrogen bonds of the Arg³⁷ side chain and the amide group are shown as light gray dotted lines, whereas intermolecular hydrogen bonds are shown in dark gray. The figure was drawn using Pymol (Version 1.2r1, Schrödinger, LLC).