Human Potassium Channel Kv Beta-subunit

Target ID:

KCNAB2B

Aliases:

AKR6A5HKvbeta2HKvbeta2.1HKvbeta2.2KCNA2BKV-BETA-2MGC117289

Deposition Date:

Wednesday 25th May 2005

Families:

Oxidoreductases

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

1ZSX

Authors:

P.WENNERSTRAND,I.JOHANNSSON,E.UGOCHUKWU,K.KAVANAGH,A.EDWARDS,C.ARROWSMITH,L.WILLIAMS,M.SUNDSTROM,K.GUO,F.VONDELFT,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

1ZSX

tabs

Structure Details

Voltage-dependent potassium channels of the Shaker or Kv1 family are critical components
in the mammalian nervous system by regulating the resting membrane potential,
frequency of action potential firing and neurotransmitter release.
Mammalian Kv1 channels are composed of four voltage-sensing, ion pore forming transmembrane Kv1 α-subunits, and four cytoplasmic β-subunits.
The diversity in Kv channel diversity arises through the combinatorial assembly of 18 α and 4 β subunit genes found in mammalian genomes.

The β-subunits are structurally related to the AKR superfamily and like these oxidoreductases adopt an (α/β)8 (TIM barrel) fold.
Despite NADP(H) binding and presence of all catalytic residues found in other AKR enzymes, thus far no physiological substrate for the β-subunits has been detected.

Materials and Methods

KCNAB2B

PDB:1ZSX

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:KCNAB2B-s001
Entry Clone Source:Origene
SGC Clone Accession:
Tag:(His 6 + TEV cleavage site): mgsshhhhhhssgrenlyfq*gh
Host:

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqghMLQFYRNLGKSGLRVSCLGLGTWVTFGGQITDEMAEQLMTLAYDNGINLFDTAEVYAAGKAEVVLGNIIKKKGWRRSSLVITTKIFWGGKAETERGLSRKHIIEGLKASLERLQLEYVDVVFANRPDPNTPMEETVRAMTHVINQGMAMYWGTSRWSSMEIMEAYSVARQFNLTPPICEQAEYHMFQREKVEVQLPELFHKIGVGAMTWSPLACGIVSGKYDSGIPPYSRASLKGYQWLKDKILSEEGRRQQAKLKELQAIAERLGCTLPQLAIAWCLRNEGVSSVLLGASNADQLMENIGAIQVLPKLSSSIIHEIDSILGNKPY
Vector:p11

Growth

Medium:
Antibiotics:
Procedure:The KCNAB2B construct was expressed in E. coli (BL21 DE3) in 1 L LB Broth in the presence of 100 Âµg/ml of ampicillin at 37 Â°C. Cells were induced at 1 mM IPTG as soon as the OD reached 0.8, and temperature was shifted to 18 Â°C, and culture was grown for 12 hrs. Cells were collected by centrifugation, and pellets were stored frozen (-20Â°C) until further use.

Purification

Buffers
Procedure
Column 1: HiTrap His

Buffers (adjusted to pH 7.5): Lysis buffer: 5mM Imidazole, 500mM NaCl, 50mM HEPES, 5%Glycerol. Wash buffer: 30mM Imidazole, 500mM NaCl, 50mM HEPES, 5%Glycerol.

Elution Buffer: 250mM Imidazole, 500mM NaCl, 5 0mM HEPES, 5% glycerol.

Procedure: Sample was loaded, washed with wash buffer and eluted in elution buffer. The collected peak was injected into size-exclusion chromatography system, and the main peak was selected for concentration using an Amicon Ultra device.

Column 2 : Superdex S200

Extraction

Buffers
Procedure
Pellets were resuspended in 25 mL lysis buffer including Protease inhibitor (complete, Roche), lysed by French Press, and the solution was centrifuged to obtain a clear supernatant (30 min, 17,000 rpm). Supernatants were processed in a 2 step chromatographic procedure using the Akta xpress (GE Healthcare) purification system.
Concentration:
Ligand
MassSpec:
Crystallization:Column 1: HiTrap His

Buffers (adjusted to pH 7.5): Lysis buffer: 5mM Imidazole, 500mM NaCl, 50mM HEPES, 5%Glycerol. Wash buffer: 30mM Imidazole, 500mM NaCl, 50mM HEPES, 5%Glycerol.

Elution Buffer: 250mM Imidazole, 500mM NaCl, 5 0mM HEPES, 5% glycerol.

Column 2 : Superdex S200
NMR Spectroscopy:
Data Collection:
Data Processing: