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Like Charge Regions (LCRs) are One of the Key Regulators of Nucleocytoplasmic Transport
510a
Wednesday, February 15, 2017
2510-Pos Board B117 Interplay Among Binding, Phosphorylation and Denaturation in Disordered 4E-BP2 as Probed by Single Molecule Fluorescence Zhenfu Zhang1, Alaji Bah2, Julie D. Forman-Kay2, Claudiu C. Gradinaru1. 1 Physics, University of Toronto, Mississauga, ON, Canada, 2The Hospital for Sick Children, Toronto, ON, Canada. Intrinsically disordered proteins (IDPs) are a class of proteins that lack well-defined 3D structures while still carry out their biological functions. They play a crucial role in mediating interactions with multiple partners and often function as protein interaction hubs. Cap-dependent initiation of translation is regulated by the interaction of the eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single molecule fluorescence resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy decay (FAD) were used to study the conformations, dynamics and binding of 4E-BP2. FAD was employed to obtain the local chain flexibility at various points within the 4E-BP2. A heterogeneous local flexibility behavior was observed throughout the protein sequence and regional amino acid composition was found to play an essential role in determining the rigidity map of the chain. The segmental flexibility is hindered in the folded region upon phosphorylation, whereas the rest of the chain becomes more flexible. The local segments become more flexible upon denaturation, which is a prominent sign of deviation from random coils. Segmental rotational correlation times and wobbling cone angles extracted for different sites along the chain provide a rigidity map of 4E-BP2 and was used to evaluate its binding mode to eIF4E. Intra-chain and inter-chain kinetics were accessed by FCS whereby heterogeneous local quenching kinetics were observed. Multi-site phosphorylation of the protein slows down the proximal chain motions and modulates the kinetics of distal regions. smFRET analysis reveals changes in the conformational ensemble in response to phosphorylation, denaturation, salt and pH. Our results demonstrate that electrostatics play a critical role in modulating the dimensions and compactness of IDPs. 2511-Pos Board B118 Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and Upon Multisite Phosphorylation Erik W. Martin1, Alex S. Holehouse2, Rohit V. Pappu2, Tanja Mittag1. 1 Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, USA, 2Biomedical Engineering and Center for Biological Systems Engineering, Washington University, St. Louis, MO, USA. Multisite phosphorylation is critical for signaling, regulation and other cellular functions and typically maps to intrinsically disordered protein regions. The accessibility of phosphorylation sites by writers, readers and erasers is presumably governed by the sequence-encoded conformational properties of the disordered regions. Expanded coil behavior, which would provide unhindered access to binding partners, has been correlated with a high fraction of charged residues. Here we interrogate the interplay of proline and charged residues in determining the global dimensions of an archetypal protein sequence undergoing multisite phosphorylation. Small-angle X-Ray scattering (SAXS), NMR and atomistic simulations were used to examine the C-terminal disordered region of the transcription factor Ash1. Despite its low FCR, unphosphorylated Ash1 is expanded and conformationally heterogeneous. Phosphorylation adds considerable charge density, but SAXS data show no perturbation to the global dimensions. However, atomistic simulations and NMR spectroscopy reveal phosphorylation-induced shifts in conformational biasing that lead to mutually compensatory local expansion and contraction, leaving the global dimensions unperturbed. Simulations of amino acid sequence variants reveal the significance of proline residues for tuning the conformational landscape. We propose a mechanism wherein synergy between proline and charged residues maintains expanded, coil-like ensembles in proteins undergoing multisite phosphorylation in all phosphorylation states. The sequence features of Ash1 are shared by other proteins; it remains to be tested whether their global dimensions are also insensitive to phosphorylation. 2512-Pos Board B119 Design of Supercharged Proteins to Impart Allosteric Behavior and their Use in Biosensing Peter Schnatz1, Joseph Brisendine1, Derek Kosciolek2, David Crouse2, Ronald Koder1. 1 City College of New York, New York, NY, USA, 2Clarkson University, Potsdam, NY, USA. We have designed a series of supercharged single-chain four-helix bundles as maquettes of intrinsically disordered proteins (IDPs). We show that a net
charge per residue above ~0.10e can impart enough electrostatic force to bias the folding equilibrium at low salt to an unfolded structure. We demonstrate that this behavior can be modulated as a function of pH and solution ionic strength, providing a wide functional dynamic range of folding energies. At the correct pH and salt concentration the proteins exhibit ligand-induced folding, and we have shown that this behavior can manifest as cooperative ligand binding. Furthermore, we are extending this supercharging to natural biopolymers, starting with green fluorescence protein, and we demonstrate that this behavior can be implanted on this fold. These adjustable characteristics have inspired a biosensing project in which we attach supercharged IDPs to a gold surface and sense conformational changes using surface plasmons. This conformational change will increase the refractive index at the gold surface, which will shift the angle of minimum reflectance. We calculate that the shift in the resonance angle caused by the ligand induced folding of an IDP is almost two orders of magnitude more than simple ligand binding to an already folded protein. Continuing reflectometry studies will provide practical insight into the use of our model as conformational switches for biosensing devices. 2513-Pos Board B120 Like Charge Regions (LCRs) are One of the Key Regulators of Nucleocytoplasmic Transport Mohaddeseh Peyro, Mohammad Soheilypour, Mohammad R.K. Mofrad. Bioengineering and Mechanical Engineering, UC Berkeley, Berkeley, CA, USA. Nuclear pore complex (NPC) is the only gateway that mediates bidirectional transport of cargos into and out of the nucleus. The NPC is made up of proteins named nucleoporins (Nups) that can be categorized into two major groups, FG Nups and non FG Nups. FG Nups are rich in Phe-Gly repeats, are intrinsically disordered and are known to be key role players in nucleocytoplasmic transport (NCT). Translocation of cargos is mediated via the transient interactions between transporters and FG Nups. Despite extensive research, the underlying mechanism of NCT through the NPC is still under much debate. Using bioinformatics techniques, we have recently discovered interesting evolutionarily conserved features in the sequences of FG Nups. One of these features is long sequences of uninterrupted positively charged residues located at the N-terminus of FG Nups that are of low charge density, which we named positive ‘‘like charge regions’’ (LCRs). Our further analysis on the biophysical role of LCRs showed that LCRs are key regulators of spatial distribution and interaction of FG Nups inside the pore. For this stage of our work, a previously developed one-bead-per-amino-acid coarse-grained molecular dynamics model was used. To further extend the model, a representation of the cargo complex was added to investigate how sequence patterns affect the interaction between cargo complex and FG Nups and how the transport process would be affected. Our results show that presence of LCRs lead to a more dynamic behavior of cargo complex. Besides, cargo complex would be able to make more interactions with different FG Nups rather than binding to one FG Nup for a longer time. This behavior would act in favor of faster transport, which is desirable for NPC as a high throughput macromolecular machine. 2514-Pos Board B121 The Intrinsically Disordered Tail of FtsZ Impacts Polymerization and Bacterial Cell Division Through Sequence-Encoded Charge Patterning Megan C. Cohan, Ammon Posey, Steven Grigsby, Alex S. Holehouse, Anuradha Mittal, Paul J. Buske, Petra Levin, Rohit V. Pappu. Washington University in St. Louis, Saint Louis, MO, USA. The sequence patterning of oppositely charged residues determines the conformational preferences polyampholytic IDRs (intrinsically disordered regions). We have used the C-terminal linker (CTL) of the FtsZ protein B. subtilis as an archetypal polyampholytic IDR to uncover the relationships between sequence-encoded interactions of IDRs and bacterial cell division. FtsZ has a tubulin-like GTPase core with a C-terminal tail (CTT) that includes a hypervariable CTL. Despite poor sequence conservation of the CTL, the parameter k that quantifies the extent segregation / mixing of oppositely charged residues is bounded between 0.15 and 0.4. Using de novo sequence design, we examined the impact of CTT k on FtsZ ring formation in vivo and the mechanisms of assembly in vitro. We find that FtsZ variants with a CTT k value within the aforementioned bounds support robust ring -formation through a GTP-dependent assembly mechanism. As k is increased further, ring formation is impaired and the CTTs promote an alternative GTP-independent assembly. Our findings provide a physical explanation for the observed bounds on k, and suggest that
Wednesday, February 15, 2017
2510-Pos Board B117 Interplay Among Binding, Phosphorylation and Denaturation in Disordered 4E-BP2 as Probed by Single Molecule Fluorescence Zhenfu Zhang1, Alaji Bah2, Julie D. Forman-Kay2, Claudiu C. Gradinaru1. 1 Physics, University of Toronto, Mississauga, ON, Canada, 2The Hospital for Sick Children, Toronto, ON, Canada. Intrinsically disordered proteins (IDPs) are a class of proteins that lack well-defined 3D structures while still carry out their biological functions. They play a crucial role in mediating interactions with multiple partners and often function as protein interaction hubs. Cap-dependent initiation of translation is regulated by the interaction of the eukaryotic initiation factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single molecule fluorescence resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy decay (FAD) were used to study the conformations, dynamics and binding of 4E-BP2. FAD was employed to obtain the local chain flexibility at various points within the 4E-BP2. A heterogeneous local flexibility behavior was observed throughout the protein sequence and regional amino acid composition was found to play an essential role in determining the rigidity map of the chain. The segmental flexibility is hindered in the folded region upon phosphorylation, whereas the rest of the chain becomes more flexible. The local segments become more flexible upon denaturation, which is a prominent sign of deviation from random coils. Segmental rotational correlation times and wobbling cone angles extracted for different sites along the chain provide a rigidity map of 4E-BP2 and was used to evaluate its binding mode to eIF4E. Intra-chain and inter-chain kinetics were accessed by FCS whereby heterogeneous local quenching kinetics were observed. Multi-site phosphorylation of the protein slows down the proximal chain motions and modulates the kinetics of distal regions. smFRET analysis reveals changes in the conformational ensemble in response to phosphorylation, denaturation, salt and pH. Our results demonstrate that electrostatics play a critical role in modulating the dimensions and compactness of IDPs. 2511-Pos Board B118 Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and Upon Multisite Phosphorylation Erik W. Martin1, Alex S. Holehouse2, Rohit V. Pappu2, Tanja Mittag1. 1 Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, USA, 2Biomedical Engineering and Center for Biological Systems Engineering, Washington University, St. Louis, MO, USA. Multisite phosphorylation is critical for signaling, regulation and other cellular functions and typically maps to intrinsically disordered protein regions. The accessibility of phosphorylation sites by writers, readers and erasers is presumably governed by the sequence-encoded conformational properties of the disordered regions. Expanded coil behavior, which would provide unhindered access to binding partners, has been correlated with a high fraction of charged residues. Here we interrogate the interplay of proline and charged residues in determining the global dimensions of an archetypal protein sequence undergoing multisite phosphorylation. Small-angle X-Ray scattering (SAXS), NMR and atomistic simulations were used to examine the C-terminal disordered region of the transcription factor Ash1. Despite its low FCR, unphosphorylated Ash1 is expanded and conformationally heterogeneous. Phosphorylation adds considerable charge density, but SAXS data show no perturbation to the global dimensions. However, atomistic simulations and NMR spectroscopy reveal phosphorylation-induced shifts in conformational biasing that lead to mutually compensatory local expansion and contraction, leaving the global dimensions unperturbed. Simulations of amino acid sequence variants reveal the significance of proline residues for tuning the conformational landscape. We propose a mechanism wherein synergy between proline and charged residues maintains expanded, coil-like ensembles in proteins undergoing multisite phosphorylation in all phosphorylation states. The sequence features of Ash1 are shared by other proteins; it remains to be tested whether their global dimensions are also insensitive to phosphorylation. 2512-Pos Board B119 Design of Supercharged Proteins to Impart Allosteric Behavior and their Use in Biosensing Peter Schnatz1, Joseph Brisendine1, Derek Kosciolek2, David Crouse2, Ronald Koder1. 1 City College of New York, New York, NY, USA, 2Clarkson University, Potsdam, NY, USA. We have designed a series of supercharged single-chain four-helix bundles as maquettes of intrinsically disordered proteins (IDPs). We show that a net
charge per residue above ~0.10e can impart enough electrostatic force to bias the folding equilibrium at low salt to an unfolded structure. We demonstrate that this behavior can be modulated as a function of pH and solution ionic strength, providing a wide functional dynamic range of folding energies. At the correct pH and salt concentration the proteins exhibit ligand-induced folding, and we have shown that this behavior can manifest as cooperative ligand binding. Furthermore, we are extending this supercharging to natural biopolymers, starting with green fluorescence protein, and we demonstrate that this behavior can be implanted on this fold. These adjustable characteristics have inspired a biosensing project in which we attach supercharged IDPs to a gold surface and sense conformational changes using surface plasmons. This conformational change will increase the refractive index at the gold surface, which will shift the angle of minimum reflectance. We calculate that the shift in the resonance angle caused by the ligand induced folding of an IDP is almost two orders of magnitude more than simple ligand binding to an already folded protein. Continuing reflectometry studies will provide practical insight into the use of our model as conformational switches for biosensing devices. 2513-Pos Board B120 Like Charge Regions (LCRs) are One of the Key Regulators of Nucleocytoplasmic Transport Mohaddeseh Peyro, Mohammad Soheilypour, Mohammad R.K. Mofrad. Bioengineering and Mechanical Engineering, UC Berkeley, Berkeley, CA, USA. Nuclear pore complex (NPC) is the only gateway that mediates bidirectional transport of cargos into and out of the nucleus. The NPC is made up of proteins named nucleoporins (Nups) that can be categorized into two major groups, FG Nups and non FG Nups. FG Nups are rich in Phe-Gly repeats, are intrinsically disordered and are known to be key role players in nucleocytoplasmic transport (NCT). Translocation of cargos is mediated via the transient interactions between transporters and FG Nups. Despite extensive research, the underlying mechanism of NCT through the NPC is still under much debate. Using bioinformatics techniques, we have recently discovered interesting evolutionarily conserved features in the sequences of FG Nups. One of these features is long sequences of uninterrupted positively charged residues located at the N-terminus of FG Nups that are of low charge density, which we named positive ‘‘like charge regions’’ (LCRs). Our further analysis on the biophysical role of LCRs showed that LCRs are key regulators of spatial distribution and interaction of FG Nups inside the pore. For this stage of our work, a previously developed one-bead-per-amino-acid coarse-grained molecular dynamics model was used. To further extend the model, a representation of the cargo complex was added to investigate how sequence patterns affect the interaction between cargo complex and FG Nups and how the transport process would be affected. Our results show that presence of LCRs lead to a more dynamic behavior of cargo complex. Besides, cargo complex would be able to make more interactions with different FG Nups rather than binding to one FG Nup for a longer time. This behavior would act in favor of faster transport, which is desirable for NPC as a high throughput macromolecular machine. 2514-Pos Board B121 The Intrinsically Disordered Tail of FtsZ Impacts Polymerization and Bacterial Cell Division Through Sequence-Encoded Charge Patterning Megan C. Cohan, Ammon Posey, Steven Grigsby, Alex S. Holehouse, Anuradha Mittal, Paul J. Buske, Petra Levin, Rohit V. Pappu. Washington University in St. Louis, Saint Louis, MO, USA. The sequence patterning of oppositely charged residues determines the conformational preferences polyampholytic IDRs (intrinsically disordered regions). We have used the C-terminal linker (CTL) of the FtsZ protein B. subtilis as an archetypal polyampholytic IDR to uncover the relationships between sequence-encoded interactions of IDRs and bacterial cell division. FtsZ has a tubulin-like GTPase core with a C-terminal tail (CTT) that includes a hypervariable CTL. Despite poor sequence conservation of the CTL, the parameter k that quantifies the extent segregation / mixing of oppositely charged residues is bounded between 0.15 and 0.4. Using de novo sequence design, we examined the impact of CTT k on FtsZ ring formation in vivo and the mechanisms of assembly in vitro. We find that FtsZ variants with a CTT k value within the aforementioned bounds support robust ring -formation through a GTP-dependent assembly mechanism. As k is increased further, ring formation is impaired and the CTTs promote an alternative GTP-independent assembly. Our findings provide a physical explanation for the observed bounds on k, and suggest that