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Long-Range Protein–Water Dynamics in Hyperactive Insect Antifreeze Proteins

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Long-Range Protein–Water Dynamics in Hyperactive Insect Antifreeze Proteins Long-range protein–water dynamics in hyperactive insect antifreeze proteins Konrad Meistera, Simon Ebbinghausa, Yao Xub, John G. Dumanc, Arthur DeVriesd, Martin Gruebelee, David M. Leitnerb, and Martina Havenitha,1 aLehrstuhl für Physikalische Chemie II, Ruhr Universität, 44801 Bochum, Germany; bDepartment of Chemistry, University of Nevada, Reno, NV 89557; cDepartment of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556; dDepartment of Animal Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and eDepartments of Chemistry and Physics, and Center of Biophysics and Computational Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved November 27, 2012 (received for review August 29, 2012) Antifreeze proteins (AFPs) are specificproteinsthatareabletolower accepted molecular mechanism can explain hysteresis at fairly the freezing point of aqueous solutions relative to the melting point. high protein concentrations, but a completely local mechanism Hyperactive AFPs, identified in insects, have an especially high ability cannot fully explain how some AFPs prevent freezing at very to depress the freezing point by far exceeding the abilities of other low concentrations, when water is present in great excess. AFPs. In previous studies, we postulated that the activity of AFPs can Therefore, the role of water molecules in the hydration shell be attributed to two distinct molecular mechanisms: (i) short-range of the AFP protein has become the focus of attention (12–15). direct interaction of the protein surface with the growing ice face Evolution has produced two structurally different solutions to and (ii) long-range interaction by protein-induced water dynamics the antifreeze problem, the AFPs and AFGPs, which have similar extending up to 20 Å from the protein surface. In the present paper, terahertz excess and antifreeze activity on a mass-scaled basis. The we combine terahertz spectroscopy and molecular simulations to latter display many saccharide OH groups in a disordered manner prove that long-range protein–water interactions make essential con- (16), whereas AFP type-1 (wfAFP-1) displays a few threonine OH tributions to the high antifreeze activity of insect AFPs from the groups projecting from one side of a relatively rigid helix. beetle Dendroides canadensis. We also support our hypothesis by In our previous study on AFGP, we proposed a distinct mech- studying the effect of the addition of the osmolyte sodium citrate. anism for their antifreeze activity: using terahertz absorption spectroscopy to probe the hydration dynamics around proteins, hydration dynamics | THz spetroscopy and thus the change in the collective water network motions, we found a considerable long-range influence on the hydration dy- namics for antifreeze-active intrinsically disordered AFGP. The ntifreezeproteins(AFPs)and antifreeze glycoproteins presence of an extended dynamic hydration shell, which is asso- (AFGPs) are classes of proteins that suppress ice growth in A ciated with a retardation in the H-bond network dynamics, was organisms and thereby enable their survival in subfreezing hab- proposed to be mandatory for its antifreeze activity (long-range itats (1). Despite their similar function, many distinct structures CHEMISTRY fi fi hypothesis) (16). Studies of the short rigid α-helix wfAFP-1 have been identi ed so far. AFPs have been identi ed in several fl organisms, including polar fish (2), insects (3), bacteria (4), and revealed that inactive AFP mutants lacked a long-range in uence plants (5). Their common characteristic is the depression of the on the H-bond dynamics (17), but the long-range effect did not freezing temperatures of ice growth of a solution without de- predict the strength of the activity. Instead, we concluded that the occurrence of antifreeze activity requires short-range OH group pressing the melting point equilibrium of protein solutions. This wf nonequilibrium phenomenon leads to a difference between the binding by AFP-1, which needs to be assisted by changes in the freezing and melting temperature, which is referred to as thermal long-range hydration dynamics (17). hysteresis (TH). TH is used as a characteristic measure for an- This conclusion is in accordance with the proposal of Sharp (9), tifreeze activity of an AFP (6). AFGPs and AFPs, as extracted who states that the recognition event of AFP is more subtle, in- from the blood of polar fish, usually exhibit up to 2° of TH activity volving more than one kind of interaction. Only then would it be fi and are termed moderately active AFPs, whereas insect AFPs can possible to combine the required high af nity (i.e., by bond using exhibit over 5° of TH and therefore, are referred to as hyperactive the OH groups in AFP) with a high selectivity, which could be AFPs. The work by Raymond and DeVries (7, 8) proposed a achieved by a significant change of the hydration dynamics around mechanism in which freezing point depression is achieved by an the ice-binding site. Retardation of the H-bond dynamics at the adsorption-inhibition mechanism, in which the proteins recognize OH binding sites could reduce the entropic cost for binding of ice and bind “quasiirreversibly” to an ice surface, thereby preventing crystals and thus, could also further assist a local mechanism. growth of ice crystals. The adsorption of the protein is thought to Together with other adaptations, such as gut voiding and prevent macroscopic ice growth in the hysteresis gap, but micro- osmolyte production, hyperactive insect AFPs allow insects to scopic growth occurs at the interface in the form of highly curved survive even at temperatures as low as 243 K (−30 °C) (1). Their fronts between adsorbed antifreeze molecules. This effect will increased hysteresis activity at a comparable binding affinity to cause a decrease of the local freezing temperature because of ice (18) must be attributed to a special enhancement effect. The the Kelvin effect, while leaving the melting temperature relatively need for a new molecular mechanism that acts at low protein unaffected (7). As recently pointed out in the work by Sharp (9), concentration is supported by the recent finding of a moderately antifreeze activity involves one of the most difficult recognition active plant AFP that binds to both basal and primary-prism planes problems in biology, the distinction between water as liquid and of ice, normally a hallmark for hyperactive insect AFPs (19). ice. The initially proposed mechanism builds on a local mech- anism. In particular, threonine (Thr) residues were proposed to play a decisive role: their hydroxyl groups were thought to be Author contributions: M.G., D.M.L., and M.H. designed research; K.M., S.E., Y.X., J.G.D., and responsible for the high affinity required for binding of the AFP A.D. performed research; J.G.D. contributed new reagents/analytic tools; K.M., S.E., Y.X., to the ice crystals. Mutations in which Thr was replaced by Ala A.D., M.G., D.M.L., and M.H. analyzed data; and K.M., Y.X., A.D., M.G., D.M.L., and M.H. were ambiguous in the conclusions that could be drawn: in some wrote the paper. cases, the mutation Thr→Ala was found to decrease TH activity The authors declare no conflict of interest. (for AFP I) (10), whereas for AFP III, the opposite mutation, This article is a PNAS Direct Submission. Ala→Thr, was found to decrease activity (11). This generally 1To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1214911110 PNAS | January 29, 2013 | vol. 110 | no. 5 | 1617–1622 Downloaded by guest on September 29, 2021 decreasing the temperature from 293 to 278 K, we find a shift of cmax from ca.12toca. 5 mg/mL. This shift indicates an increase in the size of the dynamic hydration shell for lower temperatures from ca. 20 to 27 Å (i.e., from seven to nine hydration layers). A similar behavior has been previously reported for AFGP (16). Previous CD and IR spectroscopic studies revealed that changes intemperatureofDAFP-1solutionhaveanegligibleeffecton secondary structure. Only a slight increase in the structural order and rigidity of the β-helix was found on lowering the temperature (29). Similar results were found for TmAFP and cfAFP using NMR techniques (30). Here, conserved side chains, like the thre- onine residues of the ice-binding plane, also revealed an increased rigidity when lowering the temperature (31). Simulation. To provide a more complete molecular-level picture, we have further explored the underlying molecular and hydrogen bond dynamics by accompanying molecular dynamics (MD) simulations. In Fig. 3, we compare the calculated power spectra (i.e., the predicted vibrational density of states) for water mole- cules in the hydration layer around the protein DAFP-1 and bulk water at 250 and 300 K. We observe clear differences in the Fig. 1. Model of DAFP-1 tertiary structure from D. canadensis. DAFP-1 consists β fl spectral density. At both temperatures, we predict an increase in of seven repeats, forming a -helical right-handed helix that forms a at, the density of states for water in the hydration layer compared threonine-rich ice-binding site. Positions of threonine residues are shown with bulk water in the frequency range from 2.5 to 3 THz and in red. Labels indicate positions of mutation sites. above, consistent with our experimental results. The difference between the hydration water and the bulk water spectrum is more In this study, we have focused on the investigation of a hyper- noticeable when the temperature is lowered, which is also in good active AFP from the fire-colored beetle Dendroides canadensis agreement with our experimental results. Note that 250 and 300 K (DAFP-1) as shown in Fig.
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