Abstract
Structural topology plays an important role in protein mechanical stability. Proteins with β-sandwich topology consisting of Greek key structural motifs, for example, I27 of muscle titin and 10FNIII of fibronectin, are mechanically resistant as shown by single-molecule force spectroscopy (SMFS). In proteins with β-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 β-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 β and γ-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 ∼90 pN (at a pulling speed of 1000 nm/sec), which is much lower than that of I27. Our study highlights that the β-sandwich topology proteins with a different strand-connectivity than that of I27 and 10FNIII, 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 ∼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.
Highlights
Single-molecule force spectroscopy (SMFS) studies showed that the proteins with the classical b-sandwich topology consisting of Greek key motifs in their structure are generally mechanically resistant to unfolding [1,2,3,4,5]
We demonstrate that Mcrystallin by itself is mechanically stable and provides characteristic sawtooth patterns in single-molecule pulling experiments and its mechanical stability is further enhanced upon Ca2+ binding
From the similarity in the near-UV region circular dichroism (CD) spectra of monomer and octamer it is evident that the asymmetric environment sensed by the aromatic residues is the same and the overall tertiary structure is preserved in polyprotein engineering
Summary
Single-molecule force spectroscopy (SMFS) studies showed that the proteins with the classical b-sandwich topology consisting of Greek key motifs in their structure are generally mechanically resistant to unfolding [1,2,3,4,5]. In I27, the terminal A9 and G strands are directly connected through backbone H-bonding, which is often called a ‘‘mechanical clamp’’ geometry (Figure 1, A and B), whereas 10FNIII and TNfn lack this special feature. For this reason I27 unfolds at a higher force (,200 pN) than 10FNIII and TNfn, which unfold at lower forces (,100 pN) and it was shown by experiments and simulations that the rupture of the H-bonds in the ‘‘mechanical clamp’’ of I27 leads to its mechanical unfolding [1,5,6,7,8]. The ‘‘mechanical clamp’’ geometry present in proteins with b-grasp topology is attributed to their high mechanical stability [10,11]
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