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Structural Biology of Membrane Proteins

Structural Biology of Membrane Proteins. Problems of structure determination & Membrane-specific solutions KcsA structure Mechanistic insights KvAP and voltage sensing Shaker (K V 1.2). Key Difficulties in MP Structure. Available amounts….expression systems?

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Structural Biology of Membrane Proteins

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  1. Structural Biology of Membrane Proteins • Problems of structure determination & • Membrane-specific solutions • KcsA structure • Mechanistic insights • KvAP and voltage sensing • Shaker (KV1.2)

  2. Key Difficulties in MP Structure • Available amounts….expression systems? • Solubility and solubilization; detergents • Microheterogeneity; PTMs? • Mutational alteration to facilitate crystallization; affinity tags • Homologs; genomics

  3. Channel Structures Provide.. • Details of structure • Familial relationships • Structural basis of ion selectivity • Mechanism of ion dehydration by channel • Linkage between pore and voltage sensor (for VS channels); gating mechanisms???

  4. Genomics and Channel Structures VSKC homolog (kcsA) discovered in S. lividans as ORF Monomer MW ~18kD Hydropathy analysis predicts 2 TM domains 32% identity to shaker in “signature” sequence KvAP

  5. What Defines a K+ Channel? • Founder of VS cation channel family • Widely distributed in nature; multiple isoforms in most organisms • Monomer MW ~70kD (eukaryotes) • Activated by membrane depolarization • Most isoforms have 6 predicted tm domains/monomer; functional tetramers • Mutagenic analysis of pore blockers: pore between S5 and S6

  6. KcsA: The 1st Structure Schrempf, et al (1995) “A prokaryotic potassium ion channel with two predicted transmembrane segments from S. lividans,” EMBO J 14, 5170. Cortes & Perozo (1997) “Structural dynamics of the S. lividans K channel: oligomeric stoichiometry and stability,” Biochemistry 36, 10343. Doyle et al (1998) “The structure of the potassium channel: molecular basis of K-conduction and selectivity,” Science 280, 69.

  7. VSKC Architecture?

  8. Sequences of K-channel Pore Regions

  9. Genomic Identification of KcsA Signature region

  10. Is KcsA a Channel? Single channel analysis Current-voltage relationship

  11. KcsA Oligomerizes in Detergent 1. Cell extract 2. Cell super 3. Membranes 4. DDM extract 5. NTA-purified DDM extract stain western

  12. KcsA Tetramers Maintained in DDM

  13. Structural Organization of KcsA

  14. KcsA Tetramer Defines Central Pore K+

  15. Polarity of KcsA Pore Region Blue, cationic; red, anionic; yellow, hydrophobic

  16. Aromatic Clustering at KcsA Membrane Interface

  17. Proposed Mechanism of Ion Translocation Lake Kalemia: note charge stabilization by helix dipoles

  18. Ionic Stabilization by Helix Dipole in ClC Channels

  19. Comparison of KcsA and ClC Pores ClC KcsA

  20. How Does Voltage-sensing Work? SCAM analyses consistent with transverse S4 movement

  21. The KvAP Bombshell KvAP: Structure and a Proposed Mechanism for Voltage Sensing. Jiang, MacKinnon et al (2003) “X-ray structure of a voltage-dependent K-channel,” Nature423, 33-41; “The principle of gating charge movement in a voltage-dependent K channel,” Nature423, 42-48.

  22. Sequence Homology in Voltage-dependent K-channels

  23. KvAP: a Voltage-sensitive Channel

  24. Backbone Structure of the KvAP Tetramer

  25. Putative voltage sensor paddle KvAP: The Side View

  26. Architecture of KvAP Monomer

  27. Proposed Paddle Movement during Gating

  28. Questions about the KvAP Structure • Validity of KvAP as paradigm? • Solubilization? • Modification? • Crystallization aids? • Impact of any/all on overall structure?

  29. Science, 309: 897-908

  30. Shaker and KvAP: Not quite the Same Beast

  31. Accessory Subunits in K-channels

  32. K-channel Pores Are Conserved

  33. Structure of the shaker Tetramer

  34. Backbone Comparison: KvAP vs shaker

  35. Closeup view showing the weak interaction between S4 and the pore region The Kv1.2 Tetramer: note that the nearest neighbor of S4 in the red subunit is S5 from the blue subunit Pore Communication with Voltage Sensor in Kv Channel

  36. Note S6 changes between open and closed states. Revised View of Voltage-sensing

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