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P-A3-10 Ice-binding mechanism of the antifreeze protein by MD simulations
A3 Structural dynamics P-A349 INFLUENCE PROTEASE
OF INHIBITOR DYNAMICS.
BINDING
Rln~hofcr, S.‘. Dublcr. R.‘, Kalknn, J.‘, Andrew, RUUcb, A.‘, .Scboh, D.‘, Gstach, H.‘, Stclnhaurr. H.‘. Aucr, M.‘. Ku@. A.’ ‘Inst. Thcorct. Chem., Univ. Vknaa (A); ‘Sandoz (CH);‘Sandn FoncbunSs-Inst., Vienna (A); ‘Dept. Wat~enin~en (NL)
ON
P-A3-10 ICE-BINDING MECHANISM OF THE ANTIFREEZE PROTEIN BY MD SIMULATIONS CHENG, A, MERZ, K M, Dept.of Chem.,Pennsylvania StateUniv.(U. S.A.)
HIV-1
P.‘, Vkscr, A.‘, 0.‘. Scbrelbcr, Pbarma, Ruel Bbchcm.. Univ.
Purpose: Study the structure and dynamics of the type I antifreeze protein (AFP) to understand the ice-binding mechanism. Methods: Molecular dynamics simulation to study the AFP in the gas phase, solvated by water and adsorbed on ice surfaces. Results: AFP stays as an u-helix in ail simulations. In the gas phase and the adsorbed phase, AFP bends slightly to form a salt bridge between Lysl8 and Giu22. In the soivated phase the hydrogen bonding was satisfied by the surrounding water molecules, therefore, permanent bending was not observed. Four Thr residues are on one side of the helix and the distancesbetween these residues match the ice surface lattice constant. In addition, polar side chains of Aspl, Asps, Asn16, Asn27 and Arg37 bind to the ice surface as strongly as Thr’s through hydrogen bonding. Conclusions: The polar side chains of Thr, Asn and Asp binds to the ice surface by forming hydrogen bond. The evenly spaced polar side chains match the ice lattice constant to maximize the hydrogen bonding.
Purpose: Solving the three-dimensional structure of a protein, especially when complexed with its natural ligand or inhibitors, is a major step for understanding the function of these biomolecules as well as for rational drug design. However, the impact of structural dynamics is less studied. Methods: Time-resolved fluorescence (TRF) and anisotropy data were recorded according to the single photon counting technique and were analysed with the maximum entropy method. For computer simulations the force field of the molecular dynamics (MD) package GROMOS was used, starting from the crystal structure of HIV-I protease and 2 inhibitor complexes Results: In ad&ion to overall rotation, the fluorescence anisotropy decay (FAD) is caused by tryptophan side chain dynamics. This part was computed, based on the MD simulation of the protein, and was then compared with the experimental FAD. The contributions of W6 and W42 were obtained from TRF experiments This was done for the apo-enzyme as well as for the 2 inhibitor complexes. Conclusions: We have demonstrated that binding of inhibitors to HIV-1 protease yields different FADS experimentally as well as in simulation studies Since we were able to correlate experiment and simulation we think that this dynamical approach can extend our understanding of protein function and inhibition beyond high resolution structures. This AK)
work was wpported by the OAW (APART fellowship 246 to and by the FWP (ptvject PIOSBJ-MAT to OS.).
P-A3-11 COLD-INDUCED DISSOCIATION TETRAMERIC E. C&Y TRYPTOPHANASE (Tmse) INTO DIMERS
P-A3-12 OF
T. Ben-Kasus, G. Ya. Gdalewky, R.S. Phillips. Yu. Id. To&in&y and A.H. prirola Deparbnentof Chemistryand the ks&ttes for Ap#ied Resesrch. Ben-Gudon~uni~ , k-sima. rod slid o~am;y&3 andtLhe&try, University
Purpose: Low fr uency coherent modes in *2 consuierableat&&ions due proteins have receiv to their functional impottaaee. We study the structural origins, dynamic naturq, and functional roles of low frequency modes m proteins and model s stems. Me&d: Direct detections of low frequent modes in proteins have beg limited due to weai far-infrared radiation from conventional piowbar sources. We utilized intense broad band farinfrared s nchrotron radjation as probe beam and Nicokt x 1R spectrometer f0r data collection. Results: We have measured f8r-infrared absorption spectraof oly-Gly, -Ala, -Val, -Leu, -Phe, & -T and a ew proteins at ronm and 1% K. W! found that the 109 x% bands of poiy-Leu arise from anhnrnronic c&erent mo$ons, and are characte%ic of alpha-helical su-uctures.
To study the effect of anions and cations on the dissocktion of the tetrameric tryptophanase into dimers and elucidate the nature of forces which keep the dimers together. METHOD: Dissociation was monitored by HPLC gel filtration on a Superose 12 column. RESULTS: Hoio-Tnase partialy dissociatesinto dimers in cold. Ape-Tnase almost compkteiy dissociatesat pH 7.5 in Tris-O.lM HCl or HNO3 but does not dissociate in Tris-O.iM H3PO4, 0.05M Tricine-KOH and O.lM imldazoie-HCl buffers. Yet, in imidazok-O.lM HCl, pH 7.5, 50% dissociation occurs. The W33OF mutant Tnase shows enhanced cold lability. Its incubation at OBleads to a partlai reie%seof pyridoxal-P and disso@ationinto dimers. CONCLUSION: Cold dissociation of apoTnase into dimers depends on the nature and concentration of anions. The efi%ct of diff&ent anions corresponds to their position in the Hofmeister series of salts. Our results suggest that hydrophobic ia&metiotrs play aa role in maintenance of the active structure of Tnase. PURPOSE:
42
OF INHIBITOR DYNAMICS.
BINDING
Rln~hofcr, S.‘. Dublcr. R.‘, Kalknn, J.‘, Andrew, RUUcb, A.‘, .Scboh, D.‘, Gstach, H.‘, Stclnhaurr. H.‘. Aucr, M.‘. Ku@. A.’ ‘Inst. Thcorct. Chem., Univ. Vknaa (A); ‘Sandoz (CH);‘Sandn FoncbunSs-Inst., Vienna (A); ‘Dept. Wat~enin~en (NL)
ON
P-A3-10 ICE-BINDING MECHANISM OF THE ANTIFREEZE PROTEIN BY MD SIMULATIONS CHENG, A, MERZ, K M, Dept.of Chem.,Pennsylvania StateUniv.(U. S.A.)
HIV-1
P.‘, Vkscr, A.‘, 0.‘. Scbrelbcr, Pbarma, Ruel Bbchcm.. Univ.
Purpose: Study the structure and dynamics of the type I antifreeze protein (AFP) to understand the ice-binding mechanism. Methods: Molecular dynamics simulation to study the AFP in the gas phase, solvated by water and adsorbed on ice surfaces. Results: AFP stays as an u-helix in ail simulations. In the gas phase and the adsorbed phase, AFP bends slightly to form a salt bridge between Lysl8 and Giu22. In the soivated phase the hydrogen bonding was satisfied by the surrounding water molecules, therefore, permanent bending was not observed. Four Thr residues are on one side of the helix and the distancesbetween these residues match the ice surface lattice constant. In addition, polar side chains of Aspl, Asps, Asn16, Asn27 and Arg37 bind to the ice surface as strongly as Thr’s through hydrogen bonding. Conclusions: The polar side chains of Thr, Asn and Asp binds to the ice surface by forming hydrogen bond. The evenly spaced polar side chains match the ice lattice constant to maximize the hydrogen bonding.
Purpose: Solving the three-dimensional structure of a protein, especially when complexed with its natural ligand or inhibitors, is a major step for understanding the function of these biomolecules as well as for rational drug design. However, the impact of structural dynamics is less studied. Methods: Time-resolved fluorescence (TRF) and anisotropy data were recorded according to the single photon counting technique and were analysed with the maximum entropy method. For computer simulations the force field of the molecular dynamics (MD) package GROMOS was used, starting from the crystal structure of HIV-I protease and 2 inhibitor complexes Results: In ad&ion to overall rotation, the fluorescence anisotropy decay (FAD) is caused by tryptophan side chain dynamics. This part was computed, based on the MD simulation of the protein, and was then compared with the experimental FAD. The contributions of W6 and W42 were obtained from TRF experiments This was done for the apo-enzyme as well as for the 2 inhibitor complexes. Conclusions: We have demonstrated that binding of inhibitors to HIV-1 protease yields different FADS experimentally as well as in simulation studies Since we were able to correlate experiment and simulation we think that this dynamical approach can extend our understanding of protein function and inhibition beyond high resolution structures. This AK)
work was wpported by the OAW (APART fellowship 246 to and by the FWP (ptvject PIOSBJ-MAT to OS.).
P-A3-11 COLD-INDUCED DISSOCIATION TETRAMERIC E. C&Y TRYPTOPHANASE (Tmse) INTO DIMERS
P-A3-12 OF
T. Ben-Kasus, G. Ya. Gdalewky, R.S. Phillips. Yu. Id. To&in&y and A.H. prirola Deparbnentof Chemistryand the ks&ttes for Ap#ied Resesrch. Ben-Gudon~uni~ , k-sima. rod slid o~am;y&3 andtLhe&try, University
Purpose: Low fr uency coherent modes in *2 consuierableat&&ions due proteins have receiv to their functional impottaaee. We study the structural origins, dynamic naturq, and functional roles of low frequency modes m proteins and model s stems. Me&d: Direct detections of low frequent modes in proteins have beg limited due to weai far-infrared radiation from conventional piowbar sources. We utilized intense broad band farinfrared s nchrotron radjation as probe beam and Nicokt x 1R spectrometer f0r data collection. Results: We have measured f8r-infrared absorption spectraof oly-Gly, -Ala, -Val, -Leu, -Phe, & -T and a ew proteins at ronm and 1% K. W! found that the 109 x% bands of poiy-Leu arise from anhnrnronic c&erent mo$ons, and are characte%ic of alpha-helical su-uctures.
To study the effect of anions and cations on the dissocktion of the tetrameric tryptophanase into dimers and elucidate the nature of forces which keep the dimers together. METHOD: Dissociation was monitored by HPLC gel filtration on a Superose 12 column. RESULTS: Hoio-Tnase partialy dissociatesinto dimers in cold. Ape-Tnase almost compkteiy dissociatesat pH 7.5 in Tris-O.lM HCl or HNO3 but does not dissociate in Tris-O.iM H3PO4, 0.05M Tricine-KOH and O.lM imldazoie-HCl buffers. Yet, in imidazok-O.lM HCl, pH 7.5, 50% dissociation occurs. The W33OF mutant Tnase shows enhanced cold lability. Its incubation at OBleads to a partlai reie%seof pyridoxal-P and disso@ationinto dimers. CONCLUSION: Cold dissociation of apoTnase into dimers depends on the nature and concentration of anions. The efi%ct of diff&ent anions corresponds to their position in the Hofmeister series of salts. Our results suggest that hydrophobic ia&metiotrs play aa role in maintenance of the active structure of Tnase. PURPOSE:
42