Subdomain architecture and stability of a giant repeat protein.
Tandem repeat proteins, which are widespread in the human genome, tend to exhibit high stability and favorable expression, and hence, they are emerging as promising protein scaffolds in alternative to antibodies in biotechnology. In order to investigate the origin of the stability of these proteins, we dissect the subdomain architecture of the giant repeat protein PR65/A, which comprises 15 α-helical HEAT repeats, using a series of truncations and deletions. We find that the N (HEAT 1-2) and the C (HEAT 14-15) subdomains are not capable of independent folding, but the addition of HEAT 13 to HEAT 14-15 results in an independently stable C-terminal subdomain (HEAT 13-15), which is in turn further stabilized by the inclusion of HEAT 12 (HEAT 12-15). We also further show that the stability of HEAT 13-15 is enhanced by its fusion to HEAT 1-2, and the artificial 5-HEAT-repeat protein thereby created (HEAT NC) behaves like a cooperative multidomain protein. We construct further variants, lacking one or both of the terminal subdomains, and find that such subdomains function as stabilizing caps within full-length PR65/A as in their absence, the central subdomain of the protein unfolds to form non-native β-sheet-like oligomers. Taken together, our results suggest that in full-length PR65/A, the more unstable regions within the central repeats are protected by the adjacent folded repeats, which thus act as gatekeepers by virtue of their greater stability.
Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Maksym
M
Tsytlonok
Pietro
P
Sormanni
Pamela J E
PJ
Rowling
Michele
M
Vendruscolo
Laura S
LS
Itzhaki
D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D054648:Protein Phosphatase 2 / Q000737:chemistry* / Q000378:metabolism; D058849:Protein Refolding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000737:chemistry
C512974:PPP2R2A protein, human; D012997:Solvents; D054648:Protein Phosphatase 2