Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins

BACKGROUND: Significant portion of αA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and βL-crystallin as target proteins, was increased in αA(1–172) and decreased in αA(1–168) and αA(1–162). The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins. METHODOLOGY/PRINCIPAL FINDINGS: Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and αA(1–172) was nearly the same as that of ADH and αA-wt, αA(1–168) had lower and αA(1–162) had the lowest k values. When βL-crystallin was used as the target protein, αA(1–172) had slightly higher k value than αA-wt and αA(1–168) and αA(1–162) had lower k values. As expected from earlier studies, the chaperone activity of αA(1–172) was slightly better than that of αA-wt, the chaperone activity of αA(1–168) was similar to that of αA-wt and αA(1–162) had substantially decreased chaperone activity. CONCLUSIONS/SIGNIFICANCE: Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.