Probing the three-dimensional structure of human calreticulin

Calreticulin (CRT) is an abundant soluble protein of the endoplasmic reticu lum lumen that functions as a molecular chaperone for nascent glycoproteins . We have probed the three-dimensional structure of human CRT using a serie s of biochemical and biophysical approaches in an effort to understand the molecular basis of its chaperone function. Sedimentation analysis and chemi cal cross-linking experiments showed that CRT is monodisperse and monomeric in solution with a molecular mass (MW) of 46 +/- 1 kDa. This MW value toge ther with a sedimentation coefficient, s degrees 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 consist ent with an asymmetric elongated molecule. These hydrodynamic dimensions ac count for the apparent anomalous elution position of CRT on gel filtration columns. Far-UV circular dichroism experiments showed that CRT has a cooper ative thermal denaturation transition with a midpoint temperature of 42.5 d egreesC suggesting a marginally stable structure. Proteolysis experiments s howed that the highly acidic segment at the C-terminus of CRT is most susce ptible to digest, consistent with the absence of a well-defined polypeptide backbone structure in this region of the protein. Temperature-dependent pr oteolysis with thermolysin revealed a stable core region within the N- and P-domains. A stable fragment encompassing most of the P-domain was also ide ntified 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.