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Identification and Characterization of a Repressed Troponin I-Like Epitope Structure in the C-Terminal Region of Troponin T
346a
Tuesday, February 14, 2017
constant-pH simulations that can be applied to sequences with a high content of ionizable residues. 1696-Pos Board B16 Multi-State Modeling of Biomolecules by Satisfaction of Spatial Restraints from Single-Particle Electron Microscopy Images Ilan E. Chemmama, Charles H. Greenberg, Andrej Sali. Department of Bioengineering and Therapeutic Sciences, U. of C. San Francisco, San Francisco, CA, USA. Single proteins and macromolecular complexes can exist in multiple conformational and compositional states. Accurate modeling of these ensembles of states is key to understanding and modulating the molecular function. Single-particle electron microscopy (EM) can produce a wealth of structural information. Here, we describe an integrative method for computing a multi-state model of a biomolecular system, based primarily on input particle images from EM. The approach also leverages prior knowledge about the stereochemistry of molecules, image noise, and potentially other sources of information. We validate the approach by using a synthetic benchmark, comparing it to our previous em2d method, and applying it to a real test case of g-secretase. 1697-Pos Board B17 Identification and Characterization of a Repressed Troponin I-Like Epitope Structure in the C-Terminal Region of Troponin T Taylor Heilig, J.-P. Jin. Wayne State University, Detroit, MI, USA. The troponin complex plays a central role in striated muscle contraction. Among the three subunits of troponin, troponin I (TnI) and troponin T (TnT) are encoded by genes originated from a TnI-like ancestor (Chong & Jin, J. Mol. Evol. 68:448-60, 2009). Here we report a study of an intriguing observation that the restrictive N-terminal truncation of cardiac TnT restores an epitope structure that is recognized by a monoclonal antibody (mAb) TnI-1 originally generated against a C-terminal epitope of TnI. We tested the hypothesis that this restored epitope of cardiac TnT represents an evolutionarily repressed allosteric structure. Removal of the evolutionarily added N-terminal segment of cardiac TnT removes the repression and restores a TnI-like structure in TnT, which may confer a conditional TnI-like function. Using genetically engineered TnT and fragments, we performed enzymelinked immunosorbent assay (ELISA) to assess the binding affinity of mAb TnI-1 for the restoration and localization of the TnI-like epitope. While mAb TnI-1 strongly recognizes TnI with no detectable binding to intact cardiac TnT, N-terminal truncated cardiac TnT and its T2 fragment showed significant cross reactions. ELISA titrations further showed that this repressed TnI-like epitope structure is also present in the slow and fast skeletal muscle isoforms of TnT and restorable in their T2 fragments. Since the TnI-1 epitope structure in TnI is related to a calcium-regulated tropomyosin-binding site (Zhang et al., FEBS J. 278:3348-59, 2011), the TnI-like structure restored in the C-terminal region of TnT may represent a conditional tropomyosinbinding site. Further characterizations of the function of this TnI-like tropomyosin-binding activity will provide novel insights into the structure-function relationship of TnT and the physiological significance of N-terminal modifications of TnT. 1698-Pos Board B18 NMR Studies of Conformational Selection of hnRNP H on RNA Splicing Liang-Yuan Chiu, Srinivas Penumutchu, Blanton S. Tolbert. Chemistry, Case Western Reserve University, Cleveland, OH, USA. Modularity provides proteins versatility in regulating many cellular processes. The RNA Recognition Motif (RRM) is a common protein family that adopts modularity to facilitate its biological activity. hnRNP H contains three RRMs that play important roles in RNA splicing by binding G-rich enhancer element2. However, the mechanism three RRMs use to recognize RNA is still unknown. Here, based on the analysis of paramagnetic relaxation enhancement (PRE), the data indicates that hnRNP H RRM12 adopts a closed conformation in the absence of RNA. Second, dynamics experiments, T1, T2 and NOE also reveals that RRM1 and RRM2 domain share similar motion, which suggests that compact (closed) form is major population in solution. Moreover, in closed form, one of two bindings site was buried in compact structure. Based on above observation, we propose a hypothesis that there is chemical equilibrium between open form and close form. Once hnRNP H recognizes its G-rich binding sequencing, it will release the blocking RRM from closed form and reestablish chemical equilibrium toward to open form through conformational selection in presence of RNA3. Integrated methods like NMR paramagnetic relaxation enhancement, dynamics experiments and SAXS would be utilized to address this hypothesis.
1699-Pos Board B19 Characterization of a Novel FRET Probe in a Crowded Environment using Time-Resolved Anisotropy Hannah Leopold1, Megan Currie1, Jacob Schwarz1, Arnold J. Boersma2, Erin D. Sheets1, Ahmed A. Heikal1. 1 University of Minnesota Duluth, Duluth, MN, USA, 2University of Groningen, Groningen, Netherlands. Macromolecular crowding affects numerous biophysical properties of proteins including molecular conformation, reaction kinetics and cellular localization. As a result, there is a need to quantify crowding in a heterogeneous environment both in vivo and in vitro. Recently, a novel genetically encoded FRET probe (CFP-linker-YFP) was developed as a sensor to quantitatively measure crowding in vivo and was characterized with steady-state fluorescence (Nat Meth [2015] 12:227). In this contribution, we investigated the excited state dynamics of the FRET probe in Ficoll-70 crowded environments using timeresolved fluorescence anisotropy. To differentiate between viscosity and crowding effects, similar studies were carried out in glycerol-enriched buffer (PBS, pH 7.5). Time-resolved measurements were conducted as a function of both the excitation and emission wavelengths as a means to probe the tumbling motion of the donor and acceptor separately. Our results indicate that the anisotropy of the acceptor decays as a biexponential with a fast rotational time, which is attributed to the energy transfer efficiency between the donor and acceptor. In addition, this fast, FRET-based component is sensitive to Ficoll-induced crowding in contrast with glycerol-enriched buffer. These results indicate that time-resolved anisotropy can be combined with this novel FRET probe for quantitative, non-invasive analysis of site-specific crowding. Now that a more comprehensive picture of how the FRET probe is functioning at the molecular level in vitro, further structural modifications of the probe have been carried out to increase FRET efficiency. 1700-Pos Board B20 Crytochrome and Nuclear Receptors - Expanding the Role of a Canonical Circadian Repressor Colby Sandate. The Scripps Research Institute, San Diego, CA, USA. Many organisms have evolved circadian rhythms as a molecular adaptation to the Earth’s daily light and dark cycle. Cryptochrome (CRY) is well established as the key member of the repressive arm of the mammalian molecular clock. Outside of the sleep/wake cycle, the biological clock regulates many core processes, such as metabolism. However, many of the biochemical mechanisms governing these processes have yet to be elucidated - particularly in regards to CRY repressive activity. Here, we present evidence that CRY also functions as a regulator of some nuclear receptors (NRs) - transcription factors that control the expression of myriad genes involved in metabolism, development and inflammation, among others. Using an array of biochemical techniques and electron microscopy, we aim to characterize the interaction between the NR Hepatocyte Nuclear Factor 4a (HNF4a) and CRY2 in order to shed light on CRY repressive activity in the clock and beyond. 1701-Pos Board B21 Quo Vadis, Biomacromolecular Structure Quality Radka Svobodova Varekova1, Vladimir Horsky1,2, David Sehnal1, Veronika Bendova3, Lukas Pravda1, Jaroslav Koca1. 1 Central European Institute of Technology, Brno, Czech Republic, 2Faculty of Informatics, Masaryk University, Brno, Czech Republic, 3Faculty of Science, Masaryk University, Brno, Czech Republic. Biomacromolecular structural data is one of the most interesting and important results of modern life sciences. However, all that glitters is not gold, since this treasure throve is inevitably plagued by errors and discrepancies. The issue of structure data reliability accompanies the whole process of structure discovery. It has stimulated the research community to concentrate more on data quality improvement - e.g., development of much more sophisticated data deposition workflows, highly sensitive validation tools, etc. A major issue is, how these validation efforts influence the real quality of structural data? And in general, how is structure quality changing over time and which factors affect it? Missing answers to these key questions motivated us to focus on an analysis of the current state of data quality and validation trends. Among others, we paid a special attention to ligand validation criteria as they are the most sensitive and interesting part of validation outputs. Our research has been based on validation data from our validation database ValidatorDB (http://ncbr.muni.cz/ ValidatorDB) and PDB validation reports. Furthermore, we have compiled all these validation information into the ValTrendsDB (http://ncbr.muni.cz/ ValTrendsDB). This rich database enables the whole scientific community
Tuesday, February 14, 2017
constant-pH simulations that can be applied to sequences with a high content of ionizable residues. 1696-Pos Board B16 Multi-State Modeling of Biomolecules by Satisfaction of Spatial Restraints from Single-Particle Electron Microscopy Images Ilan E. Chemmama, Charles H. Greenberg, Andrej Sali. Department of Bioengineering and Therapeutic Sciences, U. of C. San Francisco, San Francisco, CA, USA. Single proteins and macromolecular complexes can exist in multiple conformational and compositional states. Accurate modeling of these ensembles of states is key to understanding and modulating the molecular function. Single-particle electron microscopy (EM) can produce a wealth of structural information. Here, we describe an integrative method for computing a multi-state model of a biomolecular system, based primarily on input particle images from EM. The approach also leverages prior knowledge about the stereochemistry of molecules, image noise, and potentially other sources of information. We validate the approach by using a synthetic benchmark, comparing it to our previous em2d method, and applying it to a real test case of g-secretase. 1697-Pos Board B17 Identification and Characterization of a Repressed Troponin I-Like Epitope Structure in the C-Terminal Region of Troponin T Taylor Heilig, J.-P. Jin. Wayne State University, Detroit, MI, USA. The troponin complex plays a central role in striated muscle contraction. Among the three subunits of troponin, troponin I (TnI) and troponin T (TnT) are encoded by genes originated from a TnI-like ancestor (Chong & Jin, J. Mol. Evol. 68:448-60, 2009). Here we report a study of an intriguing observation that the restrictive N-terminal truncation of cardiac TnT restores an epitope structure that is recognized by a monoclonal antibody (mAb) TnI-1 originally generated against a C-terminal epitope of TnI. We tested the hypothesis that this restored epitope of cardiac TnT represents an evolutionarily repressed allosteric structure. Removal of the evolutionarily added N-terminal segment of cardiac TnT removes the repression and restores a TnI-like structure in TnT, which may confer a conditional TnI-like function. Using genetically engineered TnT and fragments, we performed enzymelinked immunosorbent assay (ELISA) to assess the binding affinity of mAb TnI-1 for the restoration and localization of the TnI-like epitope. While mAb TnI-1 strongly recognizes TnI with no detectable binding to intact cardiac TnT, N-terminal truncated cardiac TnT and its T2 fragment showed significant cross reactions. ELISA titrations further showed that this repressed TnI-like epitope structure is also present in the slow and fast skeletal muscle isoforms of TnT and restorable in their T2 fragments. Since the TnI-1 epitope structure in TnI is related to a calcium-regulated tropomyosin-binding site (Zhang et al., FEBS J. 278:3348-59, 2011), the TnI-like structure restored in the C-terminal region of TnT may represent a conditional tropomyosinbinding site. Further characterizations of the function of this TnI-like tropomyosin-binding activity will provide novel insights into the structure-function relationship of TnT and the physiological significance of N-terminal modifications of TnT. 1698-Pos Board B18 NMR Studies of Conformational Selection of hnRNP H on RNA Splicing Liang-Yuan Chiu, Srinivas Penumutchu, Blanton S. Tolbert. Chemistry, Case Western Reserve University, Cleveland, OH, USA. Modularity provides proteins versatility in regulating many cellular processes. The RNA Recognition Motif (RRM) is a common protein family that adopts modularity to facilitate its biological activity. hnRNP H contains three RRMs that play important roles in RNA splicing by binding G-rich enhancer element2. However, the mechanism three RRMs use to recognize RNA is still unknown. Here, based on the analysis of paramagnetic relaxation enhancement (PRE), the data indicates that hnRNP H RRM12 adopts a closed conformation in the absence of RNA. Second, dynamics experiments, T1, T2 and NOE also reveals that RRM1 and RRM2 domain share similar motion, which suggests that compact (closed) form is major population in solution. Moreover, in closed form, one of two bindings site was buried in compact structure. Based on above observation, we propose a hypothesis that there is chemical equilibrium between open form and close form. Once hnRNP H recognizes its G-rich binding sequencing, it will release the blocking RRM from closed form and reestablish chemical equilibrium toward to open form through conformational selection in presence of RNA3. Integrated methods like NMR paramagnetic relaxation enhancement, dynamics experiments and SAXS would be utilized to address this hypothesis.
1699-Pos Board B19 Characterization of a Novel FRET Probe in a Crowded Environment using Time-Resolved Anisotropy Hannah Leopold1, Megan Currie1, Jacob Schwarz1, Arnold J. Boersma2, Erin D. Sheets1, Ahmed A. Heikal1. 1 University of Minnesota Duluth, Duluth, MN, USA, 2University of Groningen, Groningen, Netherlands. Macromolecular crowding affects numerous biophysical properties of proteins including molecular conformation, reaction kinetics and cellular localization. As a result, there is a need to quantify crowding in a heterogeneous environment both in vivo and in vitro. Recently, a novel genetically encoded FRET probe (CFP-linker-YFP) was developed as a sensor to quantitatively measure crowding in vivo and was characterized with steady-state fluorescence (Nat Meth [2015] 12:227). In this contribution, we investigated the excited state dynamics of the FRET probe in Ficoll-70 crowded environments using timeresolved fluorescence anisotropy. To differentiate between viscosity and crowding effects, similar studies were carried out in glycerol-enriched buffer (PBS, pH 7.5). Time-resolved measurements were conducted as a function of both the excitation and emission wavelengths as a means to probe the tumbling motion of the donor and acceptor separately. Our results indicate that the anisotropy of the acceptor decays as a biexponential with a fast rotational time, which is attributed to the energy transfer efficiency between the donor and acceptor. In addition, this fast, FRET-based component is sensitive to Ficoll-induced crowding in contrast with glycerol-enriched buffer. These results indicate that time-resolved anisotropy can be combined with this novel FRET probe for quantitative, non-invasive analysis of site-specific crowding. Now that a more comprehensive picture of how the FRET probe is functioning at the molecular level in vitro, further structural modifications of the probe have been carried out to increase FRET efficiency. 1700-Pos Board B20 Crytochrome and Nuclear Receptors - Expanding the Role of a Canonical Circadian Repressor Colby Sandate. The Scripps Research Institute, San Diego, CA, USA. Many organisms have evolved circadian rhythms as a molecular adaptation to the Earth’s daily light and dark cycle. Cryptochrome (CRY) is well established as the key member of the repressive arm of the mammalian molecular clock. Outside of the sleep/wake cycle, the biological clock regulates many core processes, such as metabolism. However, many of the biochemical mechanisms governing these processes have yet to be elucidated - particularly in regards to CRY repressive activity. Here, we present evidence that CRY also functions as a regulator of some nuclear receptors (NRs) - transcription factors that control the expression of myriad genes involved in metabolism, development and inflammation, among others. Using an array of biochemical techniques and electron microscopy, we aim to characterize the interaction between the NR Hepatocyte Nuclear Factor 4a (HNF4a) and CRY2 in order to shed light on CRY repressive activity in the clock and beyond. 1701-Pos Board B21 Quo Vadis, Biomacromolecular Structure Quality Radka Svobodova Varekova1, Vladimir Horsky1,2, David Sehnal1, Veronika Bendova3, Lukas Pravda1, Jaroslav Koca1. 1 Central European Institute of Technology, Brno, Czech Republic, 2Faculty of Informatics, Masaryk University, Brno, Czech Republic, 3Faculty of Science, Masaryk University, Brno, Czech Republic. Biomacromolecular structural data is one of the most interesting and important results of modern life sciences. However, all that glitters is not gold, since this treasure throve is inevitably plagued by errors and discrepancies. The issue of structure data reliability accompanies the whole process of structure discovery. It has stimulated the research community to concentrate more on data quality improvement - e.g., development of much more sophisticated data deposition workflows, highly sensitive validation tools, etc. A major issue is, how these validation efforts influence the real quality of structural data? And in general, how is structure quality changing over time and which factors affect it? Missing answers to these key questions motivated us to focus on an analysis of the current state of data quality and validation trends. Among others, we paid a special attention to ligand validation criteria as they are the most sensitive and interesting part of validation outputs. Our research has been based on validation data from our validation database ValidatorDB (http://ncbr.muni.cz/ ValidatorDB) and PDB validation reports. Furthermore, we have compiled all these validation information into the ValTrendsDB (http://ncbr.muni.cz/ ValTrendsDB). This rich database enables the whole scientific community