The membrane proximal extracellular domain of human hGC-B folds independently.
Human Guanylyl Cyclase B (hGC-B) is a single-transmembrane receptor protein which upon binding C-type natriuretic peptide (CNP) to its extracellular domain catalyzes the intracellular conversion of GTP to the second messenger cGMP. cGMP in turn affects various physiological processes such as smooth muscle contraction, cell proliferation, phototransduction, and salt as well as fluid homeostasis. The 3-dimensional binding site of the peptide hormone is unknown, and the binding mechanism is not yet understood. Therefore, a model of the C-terminal moiety of the extracellular domain of human GC-B containing the potential binding site was derived from the crystal structure of (GC-A). The selected protein sequence was provided with an N-terminal TEV-cleavage site and fused with a 109 aa thioredoxin-tag and a hexahistidine-tag. The identity of the purified 25 kDa protein was confirmed by protein mass fingerprint and its secondary structure was determined by CD- and NMR-spectroscopy. The protein proved to be properly folded with the observed secondary structure matching the predicted secondary structure and the homologous structure in the extracellular domain of GC-A. Size exclusion chromatography confirmed the monomeric state of P-hGC-B.
Knake
Claudia
C
Department of Biopolymers and Research Center for Bio-Macromolecules, University of Bayreuth, Universitatsstrabe 30, 95440 Bayreuth, Germany.
Burmann
Björn M
BM
Schweimer
Kristian
K
Matecko
Irena
I
Rösch
Paul
P
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000302:isolation & purification; D040681:Structural Homology, Protein; D000099242:Atrial Natriuretic Factor Receptor B
D017461:Receptors, Atrial Natriuretic Factor; D000099242:Atrial Natriuretic Factor Receptor B