Spectrin domains lose cooperativity in forced unfolding.
Spectrin is a multidomain cytoskeletal protein, the component three-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies, neighboring domains of chicken brain alpha-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, nonhelical linkers (all-beta immunoglobulin domains) were used to avoid formation of nonnative helical linkers. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behavior observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behavior allows multidomain proteins to maintain integrity under applied force.
Randles
Lucy G
LG
Department of Chemistry, University of Cambridge, MRC Centre for Protein Engineering, Cambridge, United Kingdom.
Rounsevell
Ross W S
RW
Clarke
Jane
J
D003198:Computer Simulation; D064211:Connectin; D018625:Microscopy, Atomic Force; D008956:Models, Chemical; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000648:ultrastructure*; D017434:Protein Structure, Tertiary; D013049:Spectrin / Q000737:chemistry* / Q000648:ultrastructure*; D013314:Stress, Mechanical
D064211:Connectin; D009124:Muscle Proteins; D013049:Spectrin; D011494:Protein Kinases