Order in Disorder: Aggrecan CS Region Predicts a New Class of Protein Structure

Abstract

Aggrecan is a glycosylated multi‐domain protein. Secondary structure analysis of the extended CS sequence predicts no known folded motifs. It is assumed to be unstructured and its elongation caused by high glycosylation. We expressed a 31KDa CS‐peptide sequence from human aggrecan in E Coli and compared its unglycosylated structure with human aggrecan globular G3 domain.The CS‐peptide and globular G3 domain were analyzed by MALLS/SEC, SAXS, sedimentation velocity and CD (Table 1). The CS‐peptide was hydrodynamically much larger than G3 and its sedimentation coefficient 1.67S was much less than predicted (2.1S) for a random‐coil. This suggested that the CS‐peptide was very extended, and its sedimentation rate increased and CD spectrum changed in 6 M urea suggesting a significant collapse of secondary structure. Modelled as a segmented flexible chain, (Kratky/Porod and Holtzer) gave a segment length 3.3 nm, with 13 repeats and the calculated mass per unit length of 831 gmol‐1 nm‐1 was also much higher than an extended polypeptide chain (308 gmol‐1 nm‐1). Mass (Da) Mass (Da) exptl S20, W f/f0 Rh (nm) Rg (nm) G3 25700 26800 ± 700 2.38 ± 0.14 1.31 2.57 ± 0.17 N/D CS‐peptide 30827 28670 ± 1787 1.67 ± 0.11 2.01 3.99 ± 0.28 4.71 The CS‐peptide of aggrecan thus has an elongated and stiffened structure even when unglycosylated and it identifies a new class of semi‐ordered structure within disordered sequences. As 50% of all expressed protein sequences in the genome are predicted to be disordered these semi‐ordered sequences may be more commonly found than is currently recognized.