Ca2+ Binding Enhanced Mechanical Stability of an Archaeal Crystallin

Structural topology plays an important role in protein mechanical stability. Proteins with beta-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and (FNIII)-F-10 of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with beta-sandwich topology, if the terminal strands are directly connected by backbone H-bonding then this geometry can serve as a mechanical clamp''. Proteins with this geometry are shown to have very high unfolding forces. Here, we set out to explore the mechanical properties of a protein, M-crystallin, which belongs to beta-sandwich topology consisting of Greek key motifs but its overall structure lacks the mechanical clamp'' geometry at the termini. M-crystallin is a Ca2+ binding protein from Methanosarcina acetivorans that is evolutionarily related to the vertebrate eye lens beta and gamma-crystallins. We constructed an octamer of crystallin, (M-crystallin)(8), and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force similar to 90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the b-sandwich topology proteins with a different strand-connectivity than that of I27 and (FNIII)-F-10, as well as lacking mechanical clamp'' geometry, can be mechanically resistant. Furthermore, Ca2+ binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by similar to 35 pN at the same pulling speed. The differences in the mechanical properties of apo and holo M-crystallins are further characterized using pulling speed dependent measurements and they show that Ca2+ binding reduces the unfolding potential width from 0.55 nm to 0.38 nm. These results are explained using a simple two-state unfolding energy landscape.