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Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol.

Stauber M, Jakoncic J, Berger J, Karp JM, Axelbaum A, Sastow D, Buldyrev SV, Hrnjez BJ, Asherie N - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture.Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures.These findings suggest that chiral interactions are important in protein crystallization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312, USA.

ABSTRACT
Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

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The conformations of MPD from MD simulations in vacuo. (a) (R)-MPD at 300 K; (b) (R)-MPD at 370 K; (c) (S)-MPD at 300 K; (d) (S)-MPD at 370 K. In (a) the dashed lines mark the nine bins used in the free-energy calculations. The red circles are the nine locally stable conformers obtained from quantum-chemical calculations; the corresponding label for each conformer is shown in bold. The results of the simulated-annealing experiments lie within the green squares.
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fig3: The conformations of MPD from MD simulations in vacuo. (a) (R)-MPD at 300 K; (b) (R)-MPD at 370 K; (c) (S)-MPD at 300 K; (d) (S)-MPD at 370 K. In (a) the dashed lines mark the nine bins used in the free-energy calculations. The red circles are the nine locally stable conformers obtained from quantum-chemical calculations; the corresponding label for each conformer is shown in bold. The results of the simulated-annealing experiments lie within the green squares.

Mentions: The results above indicate that 1a is the most stable conformer in a variety of environments. However, the actual population of conformers in any given environment will not solely be determined by energetic factors, but also by entropic factors, i.e. by the relative free energies. To determine the relative free energies of MPD, we performed MD simulations in vacuo at 300 and 370 K for each enantiomer. The (ψ1, ψ2) conformations recorded every 1 ps during the 100 ns simulations are shown in Fig. 3 ▶. (The first 10 ns of each simulation were taken to be equilibration time and were not included in our analysis.) We see that the data cluster around the conformers examined using QC calculations (red circles in Fig. 3 ▶a). At 300 K not all of the conformers are accessible to the MD simulations, but at 370 K all nine conformers are observed for (R)-MPD and only one is not observed for (S)-MPD. Furthermore, no extraneous conformers are found, confirming that our choice of conformers for the QC calculations was reasonable. Finally, at each temperature the (R)- and (S)-MPD results show approximately the expected mirror symmetries. More specifically, for the hypothesis that the underlying distributions of the (R) and (S) conformers are the same, a χ2 test (see Supporting Information §S2) reveals that this hypothesis is accepted with p-values of greater than 0.90 (at 300 K) or 0.80 (at 370 K).


Crystallization of lysozyme with (R)-, (S)- and (RS)-2-methyl-2,4-pentanediol.

Stauber M, Jakoncic J, Berger J, Karp JM, Axelbaum A, Sastow D, Buldyrev SV, Hrnjez BJ, Asherie N - Acta Crystallogr. D Biol. Crystallogr. (2015)

The conformations of MPD from MD simulations in vacuo. (a) (R)-MPD at 300 K; (b) (R)-MPD at 370 K; (c) (S)-MPD at 300 K; (d) (S)-MPD at 370 K. In (a) the dashed lines mark the nine bins used in the free-energy calculations. The red circles are the nine locally stable conformers obtained from quantum-chemical calculations; the corresponding label for each conformer is shown in bold. The results of the simulated-annealing experiments lie within the green squares.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356360&req=5

fig3: The conformations of MPD from MD simulations in vacuo. (a) (R)-MPD at 300 K; (b) (R)-MPD at 370 K; (c) (S)-MPD at 300 K; (d) (S)-MPD at 370 K. In (a) the dashed lines mark the nine bins used in the free-energy calculations. The red circles are the nine locally stable conformers obtained from quantum-chemical calculations; the corresponding label for each conformer is shown in bold. The results of the simulated-annealing experiments lie within the green squares.
Mentions: The results above indicate that 1a is the most stable conformer in a variety of environments. However, the actual population of conformers in any given environment will not solely be determined by energetic factors, but also by entropic factors, i.e. by the relative free energies. To determine the relative free energies of MPD, we performed MD simulations in vacuo at 300 and 370 K for each enantiomer. The (ψ1, ψ2) conformations recorded every 1 ps during the 100 ns simulations are shown in Fig. 3 ▶. (The first 10 ns of each simulation were taken to be equilibration time and were not included in our analysis.) We see that the data cluster around the conformers examined using QC calculations (red circles in Fig. 3 ▶a). At 300 K not all of the conformers are accessible to the MD simulations, but at 370 K all nine conformers are observed for (R)-MPD and only one is not observed for (S)-MPD. Furthermore, no extraneous conformers are found, confirming that our choice of conformers for the QC calculations was reasonable. Finally, at each temperature the (R)- and (S)-MPD results show approximately the expected mirror symmetries. More specifically, for the hypothesis that the underlying distributions of the (R) and (S) conformers are the same, a χ2 test (see Supporting Information §S2) reveals that this hypothesis is accepted with p-values of greater than 0.90 (at 300 K) or 0.80 (at 370 K).

Bottom Line: In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture.Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures.These findings suggest that chiral interactions are important in protein crystallization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Yeshiva University, 2495 Amsterdam Avenue, New York, NY 10033-3312, USA.

ABSTRACT
Chiral control of crystallization has ample precedent in the small-molecule world, but relatively little is known about the role of chirality in protein crystallization. In this study, lysozyme was crystallized in the presence of the chiral additive 2-methyl-2,4-pentanediol (MPD) separately using the R and S enantiomers as well as with a racemic RS mixture. Crystals grown with (R)-MPD had the most order and produced the highest resolution protein structures. This result is consistent with the observation that in the crystals grown with (R)-MPD and (RS)-MPD the crystal contacts are made by (R)-MPD, demonstrating that there is preferential interaction between lysozyme and this enantiomer. These findings suggest that chiral interactions are important in protein crystallization.

Show MeSH