The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type alphaB-crystallin decreased the persistence length of the N2B-Us (from approximately 0.7 to approximately 0.2 nm) but did not alter its contour length. alphaB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 +/- 3 piconewtons); the rate constant of unfolding at zero force was estimated to be approximately 17-fold lower in the presence of alphaB-crystallin (1.4 x 10(-4) s(-1) versus 2.4 x 10(-3) s(-1)). We also found that alphaB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 +/- 6 piconewtons. The effects of alphaB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that alphaB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing alphaB-crystallin mutations suggests that the interaction between alphaB-crystallin and titin is important for normal heart function.