title: Probing the three-dimensional structure of human calreticulin.
abstract: Calreticulin (CRT) is an abundant soluble protein of the endoplasmic reticulum lumen that functions as a molecular chaperone for nascent glycoproteins. We have probed the three-dimensional structure of human CRT using a series of biochemical and biophysical approaches in an effort to understand the molecular basis of its chaperone function. Sedimentation analysis and chemical cross-linking experiments showed that CRT is monodisperse and monomeric in solution with a molecular mass (MW) of 46 +/- 1 kDa. This MW value together with a sedimentation coefficient, s(o)(20,w), of 2.71 S yielded a frictional ratio, f/f(0), of 1.65. Assuming CRT to be a prolate ellipsoid, we calculated an apparent length of 29.8 nm and diameter of 2.44 nm consistent with an asymmetric elongated molecule. These hydrodynamic dimensions account for the apparent anomalous elution position of CRT on gel filtration columns. Far-UV circular dichroism experiments showed that CRT has a cooperative thermal denaturation transition with a midpoint temperature of 42.5 degrees C suggesting a marginally stable structure. Proteolysis experiments showed that the highly acidic segment at the C-terminus of CRT is most susceptible to digest, consistent with the absence of a well-defined polypeptide backbone structure in this region of the protein. Temperature-dependent proteolysis with thermolysin revealed a stable core region within the N- and P-domains. A stable fragment encompassing most of the P-domain was also identified in the thermolytic mixture. Collectively, our results suggest that CRT is likely to be a flexible molecule in solution which may be important for its chaperone function.
authors: School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269, USA. bouvier@uconnvm.uconn.edu
authors: M
authors: M
authors: Bouvier
authors: W F
authors: WF
authors: Stafford
mesh_terms: D002135:Calcium-Binding Proteins / Q000737:chemistry*; D037282:Calreticulin; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D006358:Hot Temperature; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry*; D010446:Peptide Fragments / Q000378:metabolism; D018583:Pliability; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry*; D012996:Solutions; D013820:Thermolysin / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D002135:Calcium-Binding Proteins; D037282:Calreticulin; D003432:Cross-Linking Reagents; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D012996:Solutions; D013820:Thermolysin