The alpha-crystallins are members of the small heat shock protein (sHSP) family of molecular chaperones which have evolved to minimize intracellular protein aggregation, however they are also implicated in a number of protein deposition diseases. In this study we have employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We have replicated the oligomeric rearrangements observed for in vivo disease-related modifications, without altering the protein sequence, by refolding the alpha-crystallins in vitro. This refolding results in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of sHSPs into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behaviour associated with the alpha-crystallins in protein deposition diseases.