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 [sup.10]FNIII 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 [Ca.sup.2+] 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).sub.8], and using SMFS, we show that M-crystallin unfolds in a two-state manner with an unfolding force ~90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the [beta]- sandwich topology proteins with a different strandconnectivity than that of I27 and [sup.10]FNIII, as well as lacking "mechanical clamp" geometry, can be mechanically resistant. Furthermore, [Ca.sup.2+] binding not only stabilizes M-crystallin by 11.4 kcal/mol but also increases its unfolding force by ~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 [Ca.sup.2+] 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.