Native and recombinant proguanylin feature identical biophysical properties and are monomeric in solution.
Guanylin, an intestinal peptide hormone and endogenous ligand of guanylyl cyclase C, is produced as the corresponding prohormone proguanylin. The mature hormone consists of 15 amino acid residues, representing the COOH-terminal part of the prohormone comprised of 94 amino acid residues. Here we report the recombinant expression and purification of proguanylin with its native disulfide connectivity, as well as the biophysical characterization of the recombinant and native protein. The comparison of recombinant and native proguanylin revealed identical biophysical and structural properties, as deduced from CZE, HPLC, and mass spectrometry, as well as NMR spectroscopy and CD spectroscopy at various temperatures and pH values. Exhaustive analytical ultracentrifugation studies were employed for protein concentrations up to the millimolar range to determine the association state of recombinant as well as native proguanylin, revealing both proteins to be monomeric at the applied solution conditions. As a result, a former identified close proximity between the termini of proguanylin is due to intramolecular interactions.
Lauber
Thomas
T
Lehrstuhl für Biopolymere, Universität Bayreuth, Universitätstrasse 30, 95447 Bayreuth, Germany.
Nourse
Amanda
A
Schulz
Axel
A
Marx
Ute C
UC
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006152:Cyclic GMP / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000032:analysis; D019075:Electrophoresis, Capillary; D005768:Gastrointestinal Hormones / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D045265:Natriuretic Peptides; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry*; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions; D014461:Ultracentrifugation
D004220:Disulfides; D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012996:Solutions; C083435:preproguanylin; C072685:guanylin; D006152:Cyclic GMP