Progress 08/01/05 to 07/31/10
Outputs
OUTPUTS: This work supported Jane Vanderkooi, Bogumil Zelent, Michael Bryan, Jennifer Dashnau and Nathan Scott in studying plant antifreeze behavior. Dr. Vanderkooi is a professor at the University of Pennsylvania. Bryan is a minority post-doctoral fellow who had his own salary support, but he worked on the project and is co-author of a recent paper. Jennifer Dashnau received support from this grant when she was a graduate student; the grant provided the major part of her support. Nathan Scott received a small part of this graduate stipend when he did computational work for this project. We collaborated with Dr. Hillary Nelson and her graduate student, Laura Conlin, on the study of the effect of sugar within living yeast cells. Laura used these results for her Ph.D. thesis Our work was presented in poster form at two Department retreats and at two Departmental seminars. We presented work at CREES meetings in Washington and New Orleans. We also consulted on our work with Drs. Rick Ludescher and Srinivasan Damodaran, both awardees of CREES grants. A paper with Dr. Ludescher will be prepared. Our work is published in international journals. PARTICIPANTS: Dr. Jane Vanderkooi is a professor at the University of Pennsylvania. Michael Bryan is a minority post-doctoral fellow who had his own salary support, but he worked on the project and is co-author of a recent paper. Jennifer Dashnau was a graduate student; the grant provided the major part of her support. Nathan Scott is a graduate student and received a small part of this graduate stipend when he did computational work for this project. Dr. Hillary Nelson, Professor, was collaborator; she received no support from this grant. Laura Conlin was a graduate student collaborator on the study of the effect of sugar within living yeast cells. Laura used these results for her Ph.D. thesis She did not receive financial support from the grant. TARGET AUDIENCES: Our work was presented in poster form at two Department retreats and at two Departmental seminars. We presented work at CREES meetings in Washington and New Orleans. We also consulted on our work with Drs. Rick Ludescher and Srinivasan Damodaran, both awardees of CREES grants. A paper with Dr. Ludescher will be prepared. Our work is published in international journals. I present our work to Dr. Ludescher's group at Rutgers University. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The formation of ice crystals can kill cells, a factor that has major impact on the production of fruits and vegetables. In the original grant proposal we emphasized study of the role of glycosylation on antifreeze hysteresis activity. During the course of the research we recognized that other molecules are also as effective or more effective in preventing ice crystallization. The ability of glycerol/water mixtures to inhibit ice crystallization is linked to the concentration of glycerol and the hydrogen bonding patterns formed by these solutions. On an atomic level why glycerol prevents ice formation is clear- it breaks the 3-D arrangement of H-bonds in water. Intramolecular hydrogen bond cooperativity is associated with changes in water structure surrounding biological sugars. The OH-4 group played a pivotal role in hydration as it was able to participate in a number of hydrogen bond networks utilizing the OH-6 group. Networks that terminated within the molecule (OH-4 → OH-6 → O-5) had relatively more nonpolar-like hydration than those that ended in a free hydroxyl group. The OH-2 group modulated the strength of OH-4 networks through syndiaxial OH-2/4 intramolecular hydrogen bonding, which stabilized and induced directionality in the network. Other syndiaxial interactions only indirectly affected water structure. The results suggest that biological events such as protein-carbohydrate recognition and cryoprotection by carbohydrates may be driven by intramolecular hydrogen bond cooperativity. We examined how water is structured in yeast in stains where trehalose production is stimulated by changing growth conditions. There are subtle interactions between sugar and water. Since antifreeze glycoprotein AFGP contains the disaccharide galactose, this suggests that water interaction with the functional group is critical. Antifreeze protein from arctic insects is active in preventing ice crystallization in both the L and D stereoisometric isomers. An interesting finding is that AFP causes the melting profile to be curved. In IR spectra taken between -4 and 0 C, there is a mixture of liquid and ice water, explaining how AFP restructures water. In the original proposal we emphasized study of the role of glycosylation on antifreeze hysteresis activity. We extended this to a study of protein amino acid residues on hysteresis. Using infrared spectroscopy we examinee both solute and solvent (water). We hypothesized that groups naturally found in proteins are able to structure water strongly. These groups will have no toxicity and can be used at low concentrations, which will not alter taste or texture of food. We showed that groups that are able to structure water strongly - namely carboxylates and arginino groups - are more effective than hydroxyl groups found in sugars. Our experiments showed that the arginino group is very effective as a antifreeze agent.
Publications
- Dashnau, J. L., J. M. Vanderkooi 2007. Computational approaches to investigate how biological macromolecules can be protected in extreme conditions. J. Food Sc. 72, R1-10.
- Sharp, K.A., Vanderkooi, J. M. Acc. Chem. Res. 2010 43, 231-9. Water in the half shell: structure of water, focusing on angular structure and solvation.
- Vanderkooi, J.M., J.L. Dashnau, and B. Zelent. 2005. Temperature excursion infrared (TEIR) spectroscopy used to study hydrogen bonding between water and biomolecules. Biochim. Biophys. Acta 1749:214-233.
- Dashnau, J. L., L. K. Conlin, H.C. M. Nelson, J. M. Vanderkooi 2008. Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock. Biochim. Biophys. Acta 1780 41-50.
- Zelent B, Vanderkooi JM. Infrared spectroscopy used to study ice formation: the effect of trehalose, maltose, and glucose on melting.Anal Biochem. 2009 Jul 15;390(2):215-7.
- Zelent B, Bryan MA, Sharp KA, Vanderkooi JM. Biophys Chem. 2009 May;141(2-3):222-30. Influence of surface groups of proteins on water studied by freezing/thawing hysteresis and infrared spectroscopy.
- Pentelute, B. L., Z.P. Gates, V. Tereshko, J. L. Dashnau, J. M. Vanderkooi, A. A. Kossiakofff S. B. Kent. 2008. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers. J. Am. Chem. Soc. 130, 9695-9701.
- Pentelute, B. L., Z.P. Gates, J. L. Dashnau, J. M. Vanderkooi, A. A. Kossiakoff S. B. Kent. 2008. Mirror image forms of snow flea antifreeze protein (sfAFP) prepared by total chemical synthesis have identical antifreeze activities J. Am. Chem. Soc. 130, 9702-9707.
- Nucci, N. V., B. Zelent, J. M. Vanderkooi 2008. Pyrene-1-carboxylate in water and glycerol solutions: Origin of the change of pK upon excitation. J. Fluoresc. 18, 41-49.
- Dashnau, J.L., K.A. Sharp, and J.M. Vanderkooi. 2005. Stereochemical aspects of aldohexopyranose hydration as studied by water-water hydrogen bond angle analysis. J. Phys. Chem. 109:24152-24159.
- Dashnau, J.L., B. Zelent, and J.M. Vanderkooi. 2005. Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: Infrared and fluorescence spectroscopy and ab initio calculations. Biophys. Chem. 114:71-83.
- Thermal stability of lactoperoxidase studied by differential scanning calorimetry. Zelent B., Bryan MA and Vanderkooi, JM. Biochim. Biophys. Acta 2010 Jul;1804(7):1508-15
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Progress 08/01/06 to 07/31/07
Outputs
OUTPUTS: This grant supported the Ph.D. work of Jennifer Dashnau. She got her degree from me about 6 months ago and is now working at Merck Company. We presented our work at the Department retreat and at a Departmental seminar. We also had a poster at the American Chemical Society meeting. We attended the CREES meeting in New Orleans. Three times we visited with Prof. Ludescher at Rutgers's University for consultation. He is developing trehalose glasses for preservation of food stuffs, in work also supported by the CREES. His graduate student, Y. You, came to my laboratory twice to do experiments and the results are included in there thesis. I served on her thesis committee. The work on ATP was in collaboration with the group of Steve Kent at University of Chicago. This work will be continued. We collaborated with Dr. Hillary Nelson and her graduate student, Laura Conlin, on the study of the effect of sugar within living yeast cells. Laura used these results for her Ph.D. thesis. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: No major changes, but we will complete a study based upon our observation that peptides containing charged amino acids are especially effective in antifreeze activity.
Impacts
In this reporting cycle we were part of a group that characterized a new antifreeze protein, AFP from arctic flea. This protein is active in preventing ice crystallization in both the L and D stereoisometric isomers. This is an important control, and the results were expected since ice itself has no stereospecific forms. We have also recently explored fluorescence techniques to monitor the effect of altered water structure on proton transfer reactions. We used a compound that changes its pK upon excitation such that excitation with light causes the molecule to accept a proton. The reaction occurs as long as there is water present, but it is slowed by the presence of sugar (1). We visited Dr Richard Ludescher's laboratory at Rutgers University three times to discuss these experiments. Therefore, this method to study proton transfer in glasses may help to characterize the stability of compounds imbedded in the glass. In the original grant proposal we emphasized study of the role of glycosylation on antifreeze hysteresis activity. During the course of our work we have extended this to a study of protein amino acid residues to hysteresis. In previous work (ours and by others) hysteresis was mainly studied by observation of ice crystals using microscopy. We have now devised a new method to study protein that yields much more information about the freezing and thawing processes at the molecular level that lead to hysteresis. We examine ice formation in the infrared sample holder. By this technique we can examine both solute (substance that produces the hysteresis) and solvent (water). An interesting finding, and never reported so far in the literature, is that AFP causes the melting profile to be curved. In IR spectra taken between -4 and 0 C, we were able to show that in this region there is a mixture of liquid and ice water. This is an important point because it explains how AFP can restructure water. In another experiments ice crystals were formed at -40 C and the change in crystals after taking the sample to -4 C in the presence of AFP was then noted by observation under a microscope. The ability to re-crystallize ice - to maintain in low size crystals - is important in food preservation. We hypothesize that groups that are able to structure water strongly - namely carboxylates and arginino groups - will be even more effective than hydroxyl groups found in sugars. These groups are naturally found in proteins, and so they will have no toxicity. If they indeed turn out to be more effective, they can be used at low concentrations, which will not alter taste or texture of food. We will be examined this in the next grant period. Summary of our accomplishments from this grant and how it relates to the original grant. 1) We demonstrated how spectroscopy and computation can be used to quantitatively describe water structure around important bio-molecules and in living cells; 2) we examined the nature of water around sugar molecules and give a molecular explanation for their anti-freeze activity; 3) characterized a new AFP and hypothesized a reason for its activity.
Publications
- Pentelute, B. L., Z.P. Gates, V. Tereshko, J. L. Dashnau, J. M. Vanderkooi, A. A. Kossiakofff S. B. Kent. 2008. X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers. J. Am. Chem. Soc. 130, 9695-9701.
- Pentelute, B. L., Z.P. Gates, J. L. Dashnau, J. M. Vanderkooi, A. A. Kossiakofff S. B. Kent. 2008. Mirror image forms of snow flea antifreeze protein (sfAFP) prepared by total chemical synthesis have identical antifreeze activities J. Am. Chem. Soc. 130, 9702-9707.
- Nucci, N. V., B. Zelent, J. M. Vanderkooi 2008. Pyrene-1-carboxylate in water and glycerol solutions: Origin of the change of pK upon excitation. J. Fluoresc. 18, 41-49.
- Dashnau, J. L., J. M. Vanderkooi 2007. Computational approaches to investigate how biological macromolecules can be protected in extreme conditions. J. Food Sc. 72, R1-10.
- Dashnau, J. L., L. K. Conlin, H.C. M. Nelson, J. M. Vanderkooi 2008. Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock. BIochim. Biophys. Acta 1780 41-50.
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Progress 08/01/05 to 07/31/06
Outputs
In the past year, we have used molecular dynamics simulation and infrared spectroscopy to determine the hydrogen bond patterns first for glycerol and then for monosaccharides with water. Glycerol, a common, naturally occurring cryoprotectant, represents one of the simplest model poly-alcohols for studying how the presence of hydroxyl groups influences surrounding water structure. The ability of glycerol/water mixtures to inhibit ice crystallization is linked to the concentration of glycerol and the hydrogen bonding patterns formed by these solutions. At low glycerol concentrations, sufficient amounts of bulk-like water exist, and at low temperature, these solutions demonstrate crystallization. High glycerol concentration mixtures mimic the strong hydrogen bonding pattern seen in ice, yet crystallization does not occur. Monosaccharides present a more complex model for poly-alcohol water interactions. Like glycerol, monosaccharides contain a large number of hydroxyl
groups. Unlike the flexible molecule glycerol, monosaccharides have a more rigid structure due to their cyclization. As a result, intramolecular hydrogen bond networks are able to form to cooperatively strengthen individual hydroxyl group hydrogen bond capacity. We found that intramolecular hydrogen bond cooperativity was closely associated with changes in water structure surrounding aldohexopyranose stereoisomers. The OH-4 group played a pivotal role in hydration as it was able to participate in a number of hydrogen bond networks utilizing the OH-6 group. Networks that terminated within the molecule (OH-4 → OH-6 → O-5) had relatively more nonpolar-like hydration than those that ended in a free hydroxyl group (OH-6 → OH-4 → OH-3). The OH-2 group modulated the strength of OH-4 networks through syndiaxial OH-2/4 intramolecular hydrogen bonding, which stabilized and induced directionality in the network. The results suggest that biological events such as
protein-carbohydrate recognition and cryoprotection by carbohydrates may be driven by intramolecular hydrogen bond cooperativity. Presently, we are extending our study to include disaccharides. Trehalose, a disaccharide composed of two glucose monomers, is found in vivo to protect yeast from damages at both low and high temperature. We are using temperature-excursion infrared spectroscopy to look at how water is structured in the yeast in strains where trehalose production is stimulated. Additionally, we are using molecular dynamics simulation to understand how water is structured by both trehalose and maltose. Both disaccharides contain the same monosaccharide sub-units, yet trehalose is a better cryoprotective agent than maltose. Subtle interactions between the molecule and surrounding water are likely the cause of these differences. The study of these disaccharides will aid in interpreting the role of the sugar groups on antifreeze glycoprotein (AFGP). AFGP contains disaccharides
composed of galatose sub-units and previous work from other groups suggests a certain requirement for arrangement of these sugars in order for the protein to actively inhibit ice growth
Impacts
When an organism goes into a senescent state that enables it to survive extreme cold, heat or dehydration, it faces the same problem that we encounter in food storage. How to maintain macromolecules for long periods of time under adverse conditions? Central to this problem is to understand how protective molecules interact with water to prevent ice-induced or dehydration-induced cellular damage. Our techniques produce an advance in the study of water structure influenced by molecules. We show that simple molecules and simple arrangements of functional groups produce good results. For monosaccharides, the arrangement of functional groups rather than the chemical formula is significant for protection. Our studies also point to the reasons why the "whole" is different from the "sum of the parts" and point to rational ways to study the problem of water in food storage.
Publications
- Dashnau, J.L., Sharp, K. A. and J. M. Vanderkooi. (2005) J. Phys. Chem. 109, 24152-9. Stereochemical aspects of aldohexopyranose hydration as studied by water-water hydrogen bond angle analysis.
- Dashnau, J.L., Nucci, N.V., Sharp, K.A. and J.M. Vanderkooi, (2006) J. Phys. Chem. 110(27):13670-7. Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures.
- Dashnau, J.L. and J.M. Vanderkooi. (2006) Computational approaches to investigate how biological macromolecules can be protected in extreme conditions. In preparation. Invited review for Concise Reviews in Food Science.
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