Mechanically Unfolded Titin Immunoglobulin Domains Refold Faster and More Accurately in Presence of Chaperone Alpha-B-Crystallin

Abstract

The spring region of the giant muscle protein titin contains many immunoglobulin-like (Ig) domains, which unfold and refold under low (physiological) stretch forces. Small heat shock proteins such as alphaB-crystallin (aBC) translocate under physiological or pathological stress to the titin springs. To better understand this protective function we studied the unfolding-refolding behavior of an 8-Ig-domain titin construct (I91)8 by single-molecule AFM force spectroscopy, in the absence/presence of recombinant aBC (pH7; pH6; or pH5) or ‘control’ protein of similar size. Titin Ig domains were unfolded at 175 pN constant force applied for a variable “denature” time (tD), then the force was set to zero for a variable “quench” time (tQ) to allow for domain refolding, and finally a “probe” pulse (175 pN; tP = 5 s) was applied to test how many domains had refolded. Interestingly, Ig domain unfolding kinetics were little affected by aBC. However, upon lowering pH from 7 to 6, the refolded fraction (number of refolded Ig domains during tQ indexed to number of unfolded Ig domains during tD) decreased slightly, indicating domain misfolding. At pH5, the refolded fraction dropped by half and ∼50% of titin Ig domains showed misfolding events, an effect independent of tD (variation, 2 - 40 s). Importantly, aBC (10 µM or 20 µM) normalized the refolded fraction to values observed at pH7, whereas control protein had no such protective effect. The refolded fraction depended strongly on tQ (variation, 0.5 - 10 s), under all experimental conditions. Ig domain refolding kinetics were greatly slowed by lowering pH from 7 to 5, as quantified on refolded fraction vs. tQ plots, on which means were fit by simple exponentials. Again, aBC normalized the refolding kinetics to those observed at pH7 in the absence of aBC, whereas control protein had no such effect. We conclude that aBC speeds up titin Ig domain refolding (novel foldase activity!) and protects from domain misfolding, especially under acidic stress, which is frequently encountered in muscle cells.