Chapter 5 The Protein-Ice Interface of Anti-Freeze Molecules
5.2 Antifreeze Protein Type III
5.2.1 Deuterium Spin Lattice of Heavy Water with AFP III
Deuterium T1recovery curves of D2O with AFP III were measured at -30°C. The measurements are consistent with Siemer et al . They show that the T1 relaxation of pure D2O is 36 s while adding AFP III reduces the T1 by more than order of magnitude to 2 s. This reduction of deuterium T1 relaxation in D2O when AFP III is present is proposed to be due to proton deuterium cross relaxation. The proton T1 in AFP III is transferred via the dipolar coupling to
Figure 5.1:The line shape of (a) pure D2O and (b) D2O with 20 mg mL−1AFP III.
The temperature was calibrated to -30 °C using lead nitrate while maintaining a 12.5 kHz MAS frequency.
the deuterium in D2O ice at a protein-ice interface. The1H T1 of AFP III is shorter than the2H T1 of ice and the dipolar coupling between two types of spins results in a mixing of their T1 which means a reduction of the2H T1. This transfer is only possible if the dipolar coupling between protons in AFP III and the deuterium in the ice is strong. If there were a hydration shell with liquid water between the whole surface of the protein and the ice then the dipolar coupling would be weakened too much for this transfer of magnetisation to be observed. Hence, AFP III must have an interface with ice where the dipolar coupling between them is sufficiently strong to observe this effect on the2H T1. In figure 5.2 and table 5.1 it is observed that when the AFP III weight percentage in D2O is changed from 10 mg mL−1 to 25 mg mL−1 the T1 drops from 3 s to 0.6 s. This supports the hypothesis that AFP III is the cause of the reduction of T1 in D2O. When the weight percentage of AFP III is small the fast proton T1 is diluted among many D2O molecules and hence the ice deuterium T1 becomes an average between the D2O T1 and the AFP III T1. As the weight percentage of AFP III is increased this average is shifted towards the AFP III T1 as its proton bath begins to dominate the equilibrium. In the limit the D2O T1 would therefore become the same as the fast AFP III proton T1.
Another feature observed in the saturation recovery spectra D2O with AFP III is the presence of narrow resonance at 0 ppm. This resonance exhibits
1 0 - 6 1 0 - 5 1 0 - 4 1 0 - 3 1 0 - 2 1 0 - 1 1 0 0 1 0 1 1 0 2 1 0 3 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 D 2O L y s o z y m e I S P 1 0 m g m L - 1 I S P 1 5 m g m L - 1 I S P 2 0 m g m L - 1 I S P 2 5 m g m L - 1 s i n g l e T 1 f i t t w o T 1 f i t In te n s it y ( a rb ) R e c o v e r y T i m e ( s )
Figure 5.2: The deuterium saturation recovery T1with solid echo acquisition of
pure D2O and D2O with added AFP III at concentrations of 10, 15, 20 and 25 mg mL−1 and lysozyme at 20 mg mL−1. The temperature was calibrated to -30 °C using lead nitrate external reference and maintaining a MAS frequency of 12.5 kHz using a Bruker 4 mm HXY probe.
a different T1 compared to that of the wide quadrupolar lineshape. The former has a T1 in the order of tens of milliseconds while the latter has a T1in the order of seconds. This narrow resonance and the broad quadrupolar ice lineshape can be seen in figure 5.1b. The narrow width of this resonance implies that the D2O molecules giving rise to the resonance as well as its chemical shift is consistent with liquid water. This liquid water is thought to be in the hydration shell of the proteinas water otherwise present as supernatant has been shown to be completely frozen at -20 °C. This is consistent with the 1H spectra in figure 5.3 where there are no1H resonances present which can be assigned to the supernatant below -10 °C. The narrow resonance is present in the deuterium spectra at all weight percentages of AFP III. However, the proportion of the total magnetisation which is located in this narrow resonance increases from 7% at 10 mg mL−1 to 87% at 25 mg mL−1. This is as expected as an increase in the proportion of the AFP III would lead to an increase in the proportion of D2O present as liquid in the hydration shell. The increase in the weight percentage of AFP III appears to be linearly correlated to the proportion of magnetisation
Figure 5.3: 1H Hahn echo spectra of AFP III in H2O recorded between -10 °C and -30 °C.
in the narrow resonance which is consistent with the increase being due to the AFP III weight percentage.
Figure 5.2 shows saturation recovery curve of lysozyme, which is a non ice active protein showed an increase of the deuterium T1 to 100 s from 36 s in pure D2O as well as well as 37 of the total magnetisation being present in the narrow central resonance with a T1 of 8 ms. The increase in T1 cannot be due to the transfer of the proton T1of the protein via the dipolar coupling as the proton T1is faster than the deuterium T1. The presence of the narrow resonance previously assigned to the hydration shell is consistent with observations on AFP III. The overall behaviour of lysozyme is therefore consistent with a non ice active protein.
Table 5.1: The Deuterium T1 recovery times of D2O with antifreeze additives.
Sample Central Resonance Quadruplar Resonance T1 /ms T1/s D2O - (36±2) carrageenan - (32.5±0.8) λ-carrageenan - (39±4) ι-carrageenan (6±1) (11±1) Deltagel (4±1) (35±2) ISP (10 mg mL−1) (6±2) (2.9±0.2) ISP (15 mg mL−1) (7.5±0.6) (2±0.1) ISP (20 mg mL−1) (7±1) (1.9±0.5) ISP (25 mg mL−1) (10±3) (0.6±0.1) Lysozyme (8±2) (1.0±0.1)×102