Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We recently discovered an unusually slow reaction at room temperature that occurs on the order of days: the in crystalline reverse oxidative decay of the chemically labile (6S)-5,6,7,8-tetrahydrofolate in complex with its producing enzyme Escherichia coli dihydrofolate reductase. Here, we report the critical analysis of a representative dataset at an intermediate reaction time point. A quinonoid-like intermediate state lying between tetrahydrofolate and dihydrofolate features a near coplanar geometry of the bicyclic pterin moiety, and a tetrahedral sp3 C6 geometry is proposed based on the apparent mFo-DFc omit electron densities of the ligand. The presence of this intermediate is strongly supported by Bayesian difference refinement. Isomorphous Fo-Fo difference map and multi-state refinement analyses suggest the presence of end-state ligand populations as well, although the putative intermediate state is likely the most populated. A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin. We postulate that the constraints on the ligand imposed by the interactions with the protein environment might be the origin of the slow reaction observed by time-resolved crystallography.
{"title":"E. coli DHFR complex with a reaction intermediate","authors":"H. Cao, J. Skolnick","doi":"10.2210/PDB6MR9/PDB","DOIUrl":"https://doi.org/10.2210/PDB6MR9/PDB","url":null,"abstract":"Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We recently discovered an unusually slow reaction at room temperature that occurs on the order of days: the in crystalline reverse oxidative decay of the chemically labile (6S)-5,6,7,8-tetrahydrofolate in complex with its producing enzyme Escherichia coli dihydrofolate reductase. Here, we report the critical analysis of a representative dataset at an intermediate reaction time point. A quinonoid-like intermediate state lying between tetrahydrofolate and dihydrofolate features a near coplanar geometry of the bicyclic pterin moiety, and a tetrahedral sp3 C6 geometry is proposed based on the apparent mFo-DFc omit electron densities of the ligand. The presence of this intermediate is strongly supported by Bayesian difference refinement. Isomorphous Fo-Fo difference map and multi-state refinement analyses suggest the presence of end-state ligand populations as well, although the putative intermediate state is likely the most populated. A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin. We postulate that the constraints on the ligand imposed by the interactions with the protein environment might be the origin of the slow reaction observed by time-resolved crystallography.","PeriodicalId":48683,"journal":{"name":"Structural Dynamics-Us","volume":"6 1","pages":"024701-024701"},"PeriodicalIF":2.8,"publicationDate":"2019-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43526956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"物理与天体物理","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-11-29DOI: 10.1007/978-1-4615-0481-8_3
M. Paz
{"title":"Response of One-Degree-of-Freedom System to Harmonic Loading","authors":"M. Paz","doi":"10.1007/978-1-4615-0481-8_3","DOIUrl":"https://doi.org/10.1007/978-1-4615-0481-8_3","url":null,"abstract":"","PeriodicalId":48683,"journal":{"name":"Structural Dynamics-Us","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2018-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/978-1-4615-0481-8_3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43026584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"物理与天体物理","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-11-29DOI: 10.1007/978-1-4615-0481-8_17
M. Paz
{"title":"Dynamic Analysis of Systems with Distributed Properties","authors":"M. Paz","doi":"10.1007/978-1-4615-0481-8_17","DOIUrl":"https://doi.org/10.1007/978-1-4615-0481-8_17","url":null,"abstract":"","PeriodicalId":48683,"journal":{"name":"Structural Dynamics-Us","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2018-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46634344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"物理与天体物理","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-11-29DOI: 10.1007/978-1-4615-0481-8_7
M. Paz
{"title":"Free Vibration of a Shear Building","authors":"M. Paz","doi":"10.1007/978-1-4615-0481-8_7","DOIUrl":"https://doi.org/10.1007/978-1-4615-0481-8_7","url":null,"abstract":"","PeriodicalId":48683,"journal":{"name":"Structural Dynamics-Us","volume":" ","pages":""},"PeriodicalIF":2.8,"publicationDate":"2018-11-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46697258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"物理与天体物理","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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