Pathway shifts and thermal softening in temperature-coupled forced unfolding of spectrin domains.
Pathways of unfolding a protein depend in principle on the perturbation-whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 40-45 degrees C and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. Given the varied roles of spectrin family proteins in cell deformability, we sought to determine the coupled effects of temperature on forced unfolding. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat beta(1-4) spectrin-an alpha-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches T(m), however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. In sum, structural changes with temperature correlate with both single-molecule unfolding forces and shifts in unfolding pathways.
Biophysical Engineering Lab, Institute for Medicine and Engineering, and School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6315, USA.
Richard
R
Law
George
G
Liao
Sandy
S
Harper
Guoliang
G
Yang
David W
DW
Speicher
Dennis E
DE
Discher
D002942:Circular Dichroism; D004548:Elasticity; D006358:Hot Temperature; D018625:Microscopy, Atomic Force / Q000379:methods*; D010812:Physical Stimulation / Q000379:methods; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013049:Spectrin / Q000737:chemistry* / Q000528:radiation effects*; D013314:Stress, Mechanical; D013696:Temperature
D013049:Spectrin