Presentation on theme: "Structural Biology of Membrane Proteins Problems of structure determination & Membrane-specific solutions KcsA structure Mechanistic insights KvAP and."— Presentation transcript:
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? Solubility and solubilization; detergents Microheterogeneity; PTMs? Mutational alteration to facilitate crystallization; affinity tags Homologs; genomics
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???
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
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
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. KcsA: The 1 st Structure
How Does Voltage-sensing Work? SCAM analyses consistent with transverse S4 movement
KvAP: Structure and a Proposed Mechanism for Voltage Sensing. Jiang, MacKinnon et al (2003) “X-ray structure of a voltage-dependent K- channel,” Nature 423, 33-41; “The principle of gating charge movement in a voltage-dependent K channel,” Nature 423, 42-48. The KvAP Bombshell
Sequence Homology in Voltage-dependent K-channels
Pore Communication with Voltage Sensor in Kv Channel The Kv1.2 Tetramer: note that the nearest neighbor of S4 in the red subunit is S5 from the blue subunit Closeup view showing the weak interaction between S4 and the pore region
Revised View of Voltage-sensing Note S6 changes between open and closed states.