Mapping the electrostatic potential within the ribosomal exit tunnel.

Electrostatic potentials influence interactions among proteins and nucleic acids, the orientation of dipoles and quadrupoles, and the distribution of mobile charges. Consequently, electrostatic potentials can modulate macromolecular folding and conformational stability, as well as rates of catalysis and substrate binding. The ribosomal exit tunnel, along with its resident nascent peptide, is no less susceptible to these consequences. Yet, the electrostatics inside the tunnel have never been measured. Here we map both the electrostatic potential and accessibilities along the length of the tunnel and determine the electrostatic consequences of introducing a charged amino acid into the nascent peptide. To do this we developed novel probes and strategies. Our findings provide new insights regarding the dielectric of the tunnel and the dynamics of its local electric fields.

[1]  Z. Wang,et al.  Evolutionarily conserved features of the arginine attenuator peptide provide the necessary requirements for its function in translational regulation. , 2000, The Journal of biological chemistry.

[2]  K. Sharp,et al.  Electrostatic interactions in macromolecules: theory and applications. , 1990, Annual review of biophysics and biophysical chemistry.

[3]  A. Castellino,et al.  The design, synthesis, and evaluation of two universal doxorubicin-linkers: preparation of conjugates that retain topoisomerase II activity. , 2006, Bioorganic & medicinal chemistry letters.

[4]  C. Dobson,et al.  Three-dimensional structures of translating ribosomes by Cryo-EM. , 2004, Molecular cell.

[5]  A. Johnson,et al.  The Aqueous Pore through the Translocon Has a Diameter of 40–60 Å during Cotranslational Protein Translocation at the ER Membrane , 1997, Cell.

[6]  C. Deutsch,et al.  Secondary structure formation of a transmembrane segment in Kv channels. , 2005, Biochemistry.

[7]  C. Yanofsky,et al.  Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression. , 2005, Molecular cell.

[8]  T. Steitz,et al.  The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. , 2000, Science.

[9]  Frank Schluenzen,et al.  Structural insight into the role of the ribosomal tunnel in cellular regulation , 2003, Nature Structural Biology.

[10]  T. Steitz,et al.  The contribution of metal ions to the structural stability of the large ribosomal subunit. , 2004, RNA.

[11]  T. Steitz,et al.  The structural basis of ribosome activity in peptide bond synthesis. , 2000, Science.

[12]  J. M. Robinson,et al.  Coupled Tertiary Folding and Oligomerization of the T1 Domain of Kv Channels , 2005, Neuron.

[13]  C. Yanofsky,et al.  Reproducing tna Operon Regulation in Vitro in an S-30 System , 2001, The Journal of Biological Chemistry.

[14]  H. Bernstein,et al.  Translation arrest requires two-way communication between a nascent polypeptide and the ribosome. , 2006, Molecular cell.

[15]  P. S. Lovett,et al.  Ribosome regulation by the nascent peptide. , 1996, Microbiological reviews.

[16]  M. Ehrenberg,et al.  Regulatory Nascent Peptides in the Ribosomal Tunnel , 2002, Cell.

[17]  Barry Honig,et al.  Focusing of electric fields in the active site of Cu‐Zn superoxide dismutase: Effects of ionic strength and amino‐acid modification , 1986, Proteins.

[18]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

[19]  Guy Ziv,et al.  Ribosome exit tunnel can entropically stabilize α-helices , 2005 .

[20]  C. Stevens,et al.  Crystal structure of the tetramerization domain of the Shaker potassium channel , 1998, Nature.

[21]  A. Karlin,et al.  Substituted-cysteine accessibility method. , 1998, Methods in enzymology.

[22]  M Gerstein,et al.  The geometry of the ribosomal polypeptide exit tunnel. , 2006, Journal of molecular biology.

[23]  Jialing Lin,et al.  Both Lumenal and Cytosolic Gating of the Aqueous ER Translocon Pore Are Regulated from Inside the Ribosome during Membrane Protein Integration , 1997, Cell.

[24]  G. Reinhart,et al.  The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation , 1993, Cell.

[25]  A. Karlin,et al.  State-dependent Accessibility and Electrostatic Potential in the Channel of the Acetylcholine Receptor , 1998, The Journal of general physiology.

[26]  R. Horn,et al.  Movement and Crevices Around a Sodium Channel S3 Segment , 2002, The Journal of general physiology.

[27]  T. Rapoport,et al.  The structure of ribosome-channel complexes engaged in protein translocation. , 2000, Molecular cell.

[28]  Y. Jan,et al.  The Polar T1 Interface Is Linked to Conformational Changes that Open the Voltage-Gated Potassium Channel , 2000, Cell.

[29]  Peter J McCormick,et al.  Nascent Membrane and Secretory Proteins Differ in FRET-Detected Folding Far inside the Ribosome and in Their Exposure to Ribosomal Proteins , 2004, Cell.

[30]  C. Deutsch,et al.  Biogenesis of the T1-S1 linker of voltage-gated K+ channels. , 2007, Biochemistry.

[31]  A. Karlin,et al.  Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. , 1994, Biochemistry.

[32]  Tsung-Yu Chen,et al.  Probing the Pore of ClC-0 by Substituted Cysteine Accessibility Method Using Methane Thiosulfonate Reagents , 2003, The Journal of general physiology.

[33]  Koreaki Ito,et al.  The Ribosomal Exit Tunnel Functions as a Discriminating Gate , 2002, Cell.

[34]  J. Robinson,et al.  T1-T1 interactions occur in ER membranes while nascent Kv peptides are still attached to ribosomes. , 2001, Biochemistry.

[35]  A. Sali,et al.  Architecture of the Protein-Conducting Channel Associated with the Translating 80S Ribosome , 2001, Cell.

[36]  C. Deutsch,et al.  Pegylation: a method for assessing topological accessibilities in Kv1.3. , 2001, Biochemistry.

[37]  T. Inada,et al.  Translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization by proteasome in yeast. , 2007, Genes & development.

[38]  Peter A. Kollman,et al.  Electrostatic recognition between superoxide and copper, zinc superoxide dismutase , 1983, Nature.

[39]  G. R. Smith,et al.  The dielectric properties of water within model transbilayer pores. , 1997, Biophysical journal.

[40]  Jianli Lu,et al.  Folding zones inside the ribosomal exit tunnel , 2005, Nature Structural &Molecular Biology.

[41]  C. Deutsch,et al.  Structure Acquisition of the T1 Domain of Kv1.3 during Biogenesis , 2004, Neuron.

[42]  Entropically Driven Helix Formation , 2005, Science.

[43]  E. Campbell,et al.  Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K+ Channel , 2005, Science.

[44]  R. Horn,et al.  Probing the outer vestibule of a sodium channel voltage sensor. , 1997, Biophysical journal.