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Slow Internal Dynamics and Structural Properties of IDPs of the Ct Family: Comparing Amyloid and Non-Amyloid Variants
Saturday, February 27, 2016 signaling. Like their structured protein counterparts, IDPs can transmit the effects of binding an effector ligand at one site to another functional site, a process known as allostery. Because allostery in structured proteins has historically been interpreted in terms of propagated structural changes that are induced by effector binding, it is not clear how IDPs, lacking such welldefined structures, can allosterically affect function. Here we show mechanistically how IDPs allosterically transmit signals through a probabilistic process that originates from the simultaneous tuning of both activating and repressing ensembles of the protein, using human glucocorticoid receptor as a model. Moreover, GR modulates this signaling by producing translational isoforms with variable disordered regions. We expect this ensemble model of allostery will be important in explaining signaling in other IDPs. 17-Subg Disordered CDK Substrates act as Multi-Input Signal Processors to Control the Key Decision Points in the Cell Cycle Mart Loog. University of Tartu, Tartu, Estonia. 18-Subg Slow Internal Dynamics and Structural Properties of IDPs of the Ct Family: Comparing Amyloid and Non-Amyloid Variants Sara M. Vaiana. Arizona State University, Tempe, AZ, USA. The calcitonin peptide (Ct) family includes intrinsically disordered peptides which function as hormones (regulating glucose levels in the blood, calcium resorption in the bone, vasodilation and the transmission of pain signals, and triggering migraine attacks). They have highly conserved sequence elements, share key structural features, and bind to similar receptors. Their characterization is extremely challenging due to their low molecular weight and size, the absence of persistent secondary structure, and the high propensity of some Ct sequences to aggregate into pathological amyloid fibers. Using nanosecond laser-pump spectroscopy, in combination with other techniques, allows quantifying structural and dynamical features of these IDPs which are important for their function and pathological aggregation. 19-Subg Structural and Functional Analyses of IDPs by High-Speed AFM Imaging Toshio Ando1, Noriyuki Kodera2. 1 Physics, Kanazawa university, Kanazawa, Japan, 2Bio-AFM Center, Kanazawa university, Kanazawa, Japan. The structure of intrinsically disordered proteins (IDPs) is highly flexible and dynamically samples a multitude of conformational states. Therefore, the structural characterization of IDPs is considerably challenging. Here, we developed a single-molecule method to analyze their structure, dynamics and function, using high-speed atomic force microscopy (HS-AFM). A mechanical property of constantly or fully disordered regions within IDPs was found to be independent of their amino-acid sequences. Thanks to the invariance of this property, HSAFM imaging of IDPs allowed us to estimate the number of amino acids contained in these regions as well as to identify and characterize at the residue level other regions undergoing order-to-disorder transitions. As a test sample for functional visualization by HS-AFM, we imaged the methyl-CpG-binding protein MeCP2, whose gene mutations cause neurodevelopmental disorder called Rett syndrome. Its methyl-CpG binding domain (MBD) was observed to undergo order-to-disorder transitions, whereas other regions were mostly or constantly disordered. In MBDs of several mutated MeCP2, this two-state equilibrium was shifted to the disordered state with different degrees. This disorder propensity was found to be tightly correlated to the lower activity of MeCP2 to condense methylated dsDNA, and hence, their lower activity to condense DNA must be directly involved in causing Rett syndrome. Thus, HS-AFM imaging of single molecules opens a new opportunity to readily acquire site-resolved, quantitative and dynamic structural information on IDPs in real space as well as to study how minute changes in the structure and dynamics of IDPs are connected to diseases. 20-Subg Sequence Constraints on Folding and Binding Susan Marqusee. University of California, Berkeley, Berkeley, CA, USA. Understanding the structural and dynamic information encoded in the primary sequence of a protein is one of the most fundamental challenges in modern biology. Most studies of protein design and ligand binding are extremely native-state centric, ignoring the role of a protein’s energy landscape. The amino acid sequence of a protein, however, encodes more than the native three-dimensional structure; it encodes the entire energy landscape - an ensemble of conformations whose energetics and dynamics are finely tuned for folding, binding and activity. I will present our results demonstrating that
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for proteins without a well-folded native state, small sequence variation can have dramatic effects on ligand binding and specificity. I will also present single-molecule studies demonstrating that small changes in the sequence and environment can alter the folding trajectory of a protein.
Subgroup: Biopolymers in vivo 21-Subg Monitoring Translation in Space and Time with Ribosome Profiling Jonathan Weissman. University of California, San Francisco, San Francisco, CA, USA. 22-Subg Low Energy Barriers and a Dynamic Contact Network between Ribosomal Subunits Enable Rapid tRNA Translocation Lars V. Bock, Christian Blau, Andrea C. Vaiana, Helmut Grubmuller. Theoretical and Computational Biophysics Department, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. During protein synthesis, tRNA molecules move from the ribosome’s aminoacyl to peptidyl to exit sites, with the two ribosomal subunits remaining associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. Using molecular dynamics simulations, we here investigate conformational motions during spontaneous translocation, as well as the underlying energetics and kinetics. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and posttranslocation states. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by ‘sliding’ and ‘stepping’ mechanisms involving conserved L6, L5, and L1 residues, thus ensuring binding to the ribosome despite largescale tRNA movement through maintaining constant binding affinity. Intersubunit rotations exhibit remarkably fast intrinsic submicrosecond dynamics, which requires a fine-tuned flat free energy landscape, as any larger barrier would slow down the conformational motions. Maintaining such subtle balance between the many interactions involved is remarkable, in particular considering the large shifts the many intersubunit bridges undergo. Based on the observed occupancies of intersubunit contacts during our simulations, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The total contribution of the tRNAs -- which contact both subunits -- to the intersubunit binding enthalpy is almost constant, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation. 23-Subg Regulation of Sec-Facilitated Protein Translocation and Membrane Integration Thomas Miller. California Institute of Technology, Pasadena, CA, USA. A critical step in the biosynthesis of many proteins involves either translocation across a cellular membrane or integration into a cellular membrane. Both processes proceed via the Sec translocon - a ubiquitous and highly conserved transmembrane channel. Recent structural studies offer high-resolution snapshots of the translocon, and a wealth of biochemical and genetic data indicate important residues within the translocon; but many fundamental aspects of its mechanism and regulation remain unclear. Using both atomistic simulations and coarsegrained modeling, we investigate the conformational landscape and longtimescale dynamics of the translocon, and we explore the role of peptide substrates in the regulation of the translocation and integration pathways. Implications of these results for the regulation of Sec-mediated pathways for protein translocation, membrane integration, and integral membrane protein expression are discussed. [1] ‘‘Hydrophobically stabilized open state for the lateral gate of the Sec translocon’’ B. Zhang and T. F. Miller III, Proc. Nat. Acad. Sci. USA, 107, 5399 (2010). [2] ‘‘Direct simulation of early-stage Sec-facilitated protein translocation’’ B. Zhang and T. F. Miller III, J. Am. Chem. Soc., 134, 13700 (2012). [3] ‘‘Long-timescale dynamics and the regulation of Sec-facilitated protein translocation’’ B. Zhang and T. F. Miller III, Cell Rep., 2, 927 (2012). [4] ‘‘Regulation of multispanning membrane protein topology via posttranslational annealing’’ R. C. Van Lehn, B. Zhang, and T.F. Miller III, eLife, in press (2015). http://dx. doi.org/10.7554/eLife.08697.
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for proteins without a well-folded native state, small sequence variation can have dramatic effects on ligand binding and specificity. I will also present single-molecule studies demonstrating that small changes in the sequence and environment can alter the folding trajectory of a protein.
Subgroup: Biopolymers in vivo 21-Subg Monitoring Translation in Space and Time with Ribosome Profiling Jonathan Weissman. University of California, San Francisco, San Francisco, CA, USA. 22-Subg Low Energy Barriers and a Dynamic Contact Network between Ribosomal Subunits Enable Rapid tRNA Translocation Lars V. Bock, Christian Blau, Andrea C. Vaiana, Helmut Grubmuller. Theoretical and Computational Biophysics Department, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. During protein synthesis, tRNA molecules move from the ribosome’s aminoacyl to peptidyl to exit sites, with the two ribosomal subunits remaining associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. Using molecular dynamics simulations, we here investigate conformational motions during spontaneous translocation, as well as the underlying energetics and kinetics. Resolving fast transitions between states, we find that tRNA motions govern the transition rates within the pre- and posttranslocation states. The L1 stalk drives the tRNA from the peptidyl site and links intersubunit rotation to translocation. Displacement of tRNAs is controlled by ‘sliding’ and ‘stepping’ mechanisms involving conserved L6, L5, and L1 residues, thus ensuring binding to the ribosome despite largescale tRNA movement through maintaining constant binding affinity. Intersubunit rotations exhibit remarkably fast intrinsic submicrosecond dynamics, which requires a fine-tuned flat free energy landscape, as any larger barrier would slow down the conformational motions. Maintaining such subtle balance between the many interactions involved is remarkable, in particular considering the large shifts the many intersubunit bridges undergo. Based on the observed occupancies of intersubunit contacts during our simulations, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The total contribution of the tRNAs -- which contact both subunits -- to the intersubunit binding enthalpy is almost constant, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation. 23-Subg Regulation of Sec-Facilitated Protein Translocation and Membrane Integration Thomas Miller. California Institute of Technology, Pasadena, CA, USA. A critical step in the biosynthesis of many proteins involves either translocation across a cellular membrane or integration into a cellular membrane. Both processes proceed via the Sec translocon - a ubiquitous and highly conserved transmembrane channel. Recent structural studies offer high-resolution snapshots of the translocon, and a wealth of biochemical and genetic data indicate important residues within the translocon; but many fundamental aspects of its mechanism and regulation remain unclear. Using both atomistic simulations and coarsegrained modeling, we investigate the conformational landscape and longtimescale dynamics of the translocon, and we explore the role of peptide substrates in the regulation of the translocation and integration pathways. Implications of these results for the regulation of Sec-mediated pathways for protein translocation, membrane integration, and integral membrane protein expression are discussed. [1] ‘‘Hydrophobically stabilized open state for the lateral gate of the Sec translocon’’ B. Zhang and T. F. Miller III, Proc. Nat. Acad. Sci. USA, 107, 5399 (2010). [2] ‘‘Direct simulation of early-stage Sec-facilitated protein translocation’’ B. Zhang and T. F. Miller III, J. Am. Chem. Soc., 134, 13700 (2012). [3] ‘‘Long-timescale dynamics and the regulation of Sec-facilitated protein translocation’’ B. Zhang and T. F. Miller III, Cell Rep., 2, 927 (2012). [4] ‘‘Regulation of multispanning membrane protein topology via posttranslational annealing’’ R. C. Van Lehn, B. Zhang, and T.F. Miller III, eLife, in press (2015). http://dx. doi.org/10.7554/eLife.08697.